Replication defective adenovirus vector in vaccination

ABSTRACT

Methods for generating immune responses using adenovirus vectors that allow multiple vaccinations with the same adenovirus vector and vaccinations in individuals with preexisting immunity to adenovirus are provided.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application Nos.61/693,187, filed Aug. 24, 2012, 61/694,013, filed Aug. 28, 2012,61/748,494, filed Jan. 3, 2013, and 61/756,870, filed Jan. 25, 2013 andwhich applications are herein incorporated by reference in theirentirety and to which applications we claim priority under 35 USC §120.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Contract No. HHSN261200900059C, awarded by the National Cancer Institute; and ContractNo. HHSN 261201100097C, awarded by the National Cancer Institute; GrantNo. 1R43CA134063, awarded by the National Cancer Institute; Grant No.2R44CA134063 awarded by the National Cancer Institute; and Contract No.HHSN 261200900059C, awarded by the National Cancer Institute; andContract No. HHSN 261201100097C, awarded by the National CancerInstitute. The government has certain rights in the invention.

BACKGROUND

Cancer immunotherapy achieved by delivering tumor-associated antigens(TAA) has recently demonstrated survival benefits; however limitationsto these strategies exist and more immunologically potent vaccines areneeded. To address the low immunogenicity of self-tumor antigens, avariety of advanced, multi-component vaccination strategies includingco-administration of adjuvants and immune stimulating cytokines havebeen employed. Alternatives include the use of recombinant viral vectorsthat inherently provide innate pro-inflammatory signals, whilesimultaneously engineered to express the antigen of interest. Ofparticular interest are adenovirus serotype-5 (Ad5)-basedimmunotherapeutics that have been repeatedly used in humans to inducerobust T cell-mediated immune (CMI) responses, all while maintaining anextensive safety profile. In addition, Ad5 vectors can be reliablymanufactured in large quantities and are stable for storage and deliveryfor outpatient administration. Nonetheless, a major obstacle to the useof first generation (E1-deleted) Ad5-based vectors is the high frequencyof pre-existing anti-adenovirus type 5 neutralizing antibodies. Theseantibodies can be present in a potential vaccinee due to either priorwild type adenovirus infection and/or induction of adenovirusneutralizing antibodies by repeated injections with Ad5-based vaccines,each resulting in inadequate immune stimulation against the target TAA.

A major problem with adenovirus vectors has been their inability tosustain long-term transgene expression due largely to the host immuneresponse that eliminates the adenovirus vector and virally transducedcells in immune-competent subjects. Thus, the use of First Generationadenovirus vector vaccines is severely limited by preexisting or inducedimmunity of vaccines to adenovirus (Ad) (Yang, et al. J Virol 77/799-803(2003); Casimiro, et al. J Virol 77/6305-6313 (2003)). One groupreported that a preponderance of humans have antibody against adenovirustype 5 (Ad5), the most widely used serotype for gene transfer vectors,and that two-thirds of humans studied have lympho-proliferativeresponses against Ad (Chirmule, et al. Gene Ther 6/1574-1583 (1999)). Inanother study, an adenovirus vector vaccine carrying an HIV-1 envelopegene was incapable of reimmunizing a primed immune response usingnon-adjuvanted DNA (Barouch, et al. J. Virol 77/8729-8735 (2003)).Another group reported that non-human primates having pre-existingimmunity against Ad5 due to a single immunization with Ad5 were unableto generate transgene-specific antibodies to HIV proteins, as well asaltering the overall T cell responses (McCoy, et al. J. Virol81/6594-6604 (2007)).

There are numerous mechanisms by which preexisting immunity interfereswith adenovirus vector vaccines but one major concern is the presence ofneutralizing antibody followed by cell mediated immune elimination of Adinfected antigen harboring cells. Both of these responses can bedirected to several Ad proteins. One approach is to increase the vectorvaccine dose. Although there is evidence that increasing vaccine dosescan increase induction of desired cell mediated immune (CMI) responsesin Ad-immune animals (Barouch, et al. J. Virol 77/8729-8735 (2003)), itoften results in unacceptable adverse effects in animals and humans.When using First Generation Ad5 vector vaccines, one option can be touse the approach of a heterologous prime-boost regimen, using naked(non-vectored) DNA as the priming vaccination, followed by an Ad5 vectorimmunization. This protocol may result in a subsequent immune responseagainst Ad5 such that one cannot administer a further re-immunization(boost) with the same (or a different) adenovirus vector vaccine thatutilizes the same viral backbone. Therefore, with the current FirstGeneration of Ad5 vectors, using this approach can also abrogate anyfurther use of Ad5 vector immunization in the Ad5 immunized vaccinee.

First Generation (E1 deleted) adenovirus vector vaccines express Ad lategenes, albeit at a decreased level and over a longer time period thanwild-type Ad virus (Nevins, et al. Cell 26/213-220 (1981); Gaynor, etal. Cell 33/683-693 (1983); Yang, et al. J Virol 70/7209-7212 (1996)).When using First Generation adenovirus vectors for immunization, vaccineantigens are presented to the immune system simultaneously with highlyimmunogenic Ad capsid proteins. The major problem with these adenovirusvectors is that the immune responses generated are less likely to bedirected to the desired vaccine epitopes (McMichael, et al. Nat RevImmunol 2/283-291 (2002)) and more likely to be directed to theadenovirus-derived antigens, i.e., antigenic competition. There iscontroversy about the mechanism by which First Generation adenovirusvectors are potent immunogens. It has been hypothesized that thecomposition of the Ad capsid or a toxic effect of viral genes createsgeneralized inflammation resulting in a nonspecific immune stimulatoryeffect. The E1 proteins of Ad act to inhibit inflammation followinginfection (Schaack, et al. PNAS 101/3124-3129 (2004)). Removal of thegene segments for these proteins, which is the case for First Generationadenovirus vectors, results in increased levels of inflammation(Schaack, et al. PNAS 101/3124-3129 (2004); Schaack, et al. ViralImmunol 18/79-88 (2005)).

Thus, it is apparent that there remains a need for a more effectivecancer vaccine vector candidate. In particular, there remains a need inthe art for cancer targeting Ad vaccine vectors that allow multiplevaccinations and vaccinations in individuals with preexisting immunityto Ad. The present invention provides this and other advantages.

BRIEF SUMMARY

In a first aspect, the invention relates to a composition comprising arecombinant nucleic acid vector comprising a sequence with a firstidentity value of at least 80% to SEQ ID NO: 3. In some embodiments, therecombinant nucleic acid vector further comprises a region with a secondidentity value of at least 80% to a region in SEQ ID NO: 3, wherein theregion is selected from 26048-26177, 26063-26141, 1-103, 54-103,32214-32315, and 32214-32262. In some embodiments, the recombinantnucleic acid vector further comprises a region encoding a peptide with athird identity value of at least 80% to a peptide encoded by a region inSEQ ID NO: 3 between positions 1057 and 3165. In some embodiments, thefirst identity value is at least 90%. In some embodiments, the firstidentity value is at least 95%. In some embodiments, the first identityvalue is at least 99%. In some embodiments, the first identity value is100%. In some embodiments, the second identity value is at least 90%. Insome embodiments, the second identity value is at least 95%. In someembodiments, the second identity value is at least 99%. In someembodiments, the second identity value is 100%. In some embodiments, thethird identity value is at least 90%. In some embodiments, the thirdidentity value is at least 95%. In some embodiments, the third identityvalue is at least 99%. In some embodiments, the third identity value is100%.

In a second aspect, the invention relates to a composition comprising arecombinant nucleic acid vector comprising a sequence encoding amodified CEA; wherein the modified CEA comprises a sequence with a firstidentity value of at least 80% to SEQ ID NO: 2; and wherein therecombinant nucleic acid vector comprises a replication defectiveadenovirus vector. In some embodiments, the replication defectiveadenovirus vector comprises a replication defective adenovirus 5 vector.In some embodiments, the replication defective adenovirus vectorcomprises a deletion in the E2b region. In some embodiments, thereplication defective adenovirus vector further comprises a deletion inthe E1 region. In some embodiments, the first identity value is at least90%. In some embodiments, the first identity value is at least 95%. Insome embodiments, the first identity value is at least 99%. In someembodiments, the first identity value is 100%. In some embodiments, thefirst identity value is at least 90%. In some embodiments, the firstidentity value is at least 95%. In some embodiments, the first identityvalue is at least 99%. In some embodiments, the first identity value is100%. In some embodiments, the first identity value is at least 90%. Insome embodiments, the first identity value is at least 95%. In someembodiments, the first identity value is at least 99%. In someembodiments, the first identity value is 100%.

In a third aspect, the invention relates to a composition comprising arecombinant nucleic acid vector comprising a sequence encoding amodified CEA; wherein the modified CEA comprises a modification in up to25 amino acids; and wherein the recombinant nucleic acid vectorcomprises a replication defective adenovirus 5 vector having a deletionin the E2b region. In some embodiments, the modified CEA comprises amodification in up to 20 amino acids. In some embodiments, the modifiedCEA comprises a modification in up to 15 amino acids. In someembodiments, the modified CEA comprises a modification in up to 10 aminoacids. In some embodiments, the modified CEA comprises a modification inup to 5 amino acids. In some embodiments, the recombinant nucleic acidvector is capable of effecting overexpression of the modified CEA intransfected cells. In some embodiments, the recombinant nucleic acidvector is capable of inducing a specific immune response against cellsexpressing CEA in a human that is at least 25 fold over basal. In someembodiments, the human has an inverse Ad5 neutralizing antibody titer ofgreater than 200. In some embodiments, the human has an inverse Ad5neutralizing antibody titer of greater than 4767. In some embodiments,the immune response is measured as CEA antigen specific cell-mediatedimmunity (CMI). In some embodiments, the immune response is measured asCEA antigen specific IFN-γ secretion. In some embodiments, the immuneresponse is measured as CEA antigen specific IL-2 secretion. In someembodiments, the immune response against CEA is measured by ELISspotassay. In some embodiments, the CEA antigen specific CMI is greater than300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral blood mononuclearcells (PBMC). In some embodiments, the immune response is measured by Tcell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic CEAexpressing cells from a tumor cell line or from an autologous tumor. Insome embodiments, the composition further comprises an immunogeniccomponent. In some embodiments, the immunogenic component comprises acytokine selected from the group of. IFN-γ, TNFα IL-2, IL-8, IL-12,IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In someembodiments, the immunogenic component is selected from the groupconsisting of IL-7, a nucleic acid encoding IL-7, a protein withsubstantial identity to IL-7, and a nucleic acid encoding a protein withsubstantial identity to IL-7. In some embodiments, the modified CEAcomprises a modification in 1 amino acid compared to wild type. In someembodiments, the modification comprises a substitution into aspartate ina position corresponding to position 610 in SEQ. ID. NO. 3.

In a fourth aspect, the invention relates to a composition comprising arecombinant nucleic acid vector, wherein the recombinant nucleic acidvector comprises a replication defective adenovirus vector, and whereinupon administration to a human, the composition is capable of inducingan immune response directed towards cells expressing CEA antigen in saidhuman; wherein the immune response comprises cell mediated immunity. Insome embodiments, the replication defective adenovirus vector comprisesa replication defective adenovirus 5 vector. In some embodiments, theimmune response is measured as CEA antigen specific cell-mediatedimmunity (CMI). In some embodiments, the immune response is measured asCEA antigen specific IFN-γ secretion. In some embodiments, the immuneresponse is measured as CEA antigen specific IL-2 secretion. In someembodiments, the immune response against CEA is measured by ELISspotassay. In some embodiments, the CEA antigen specific CMI is greater than300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral blood mononuclearcells (PBMC). In some embodiments, the immune response is measured by Tcell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic CEAexpressing cells from a tumor cell line or from an autologous tumor.

In a fifth aspect, the invention relates to a vial comprising acomposition consisting of a therapeutic solution of a volume in therange of 0.8-1.2 ml, the therapeutic solution comprising 4.5-5.5×10¹¹virus particles; wherein the virus particle is a replication defectiveadenovirus comprising a recombinant nucleic acid vector. In someembodiments, the recombinant nucleic acid vector is capable of effectingoverexpression of the modified CEA in transfected cells. In someembodiments, the replication defective adenovirus comprises a nucleicacid sequence encoding a protein that is capable of inducing a specificimmune response against CEA expressing cells in a human. In someembodiments, the replication defective adenovirus vector comprises areplication defective adenovirus 5 vector. In some embodiments, theimmune response is measured as CEA antigen specific cell-mediatedimmunity (CMI). In some embodiments, the immune response is measured asCEA antigen specific IFN-γ secretion. In some embodiments, the immuneresponse is measured as CEA antigen specific IL-2 secretion. In someembodiments, the immune response against CEA is measured by ELISspotassay. In some embodiments, the CEA antigen specific CMI is greater than300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral blood mononuclearcells (PBMC). In some embodiments, the immune response is measured by Tcell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic CEAexpressing cells from a tumor cell line or from an autologous tumor. Insome embodiments, the therapeutic solution comprises 4.8-5.2×10¹¹ virusparticles. In some embodiments, the therapeutic solution comprises4.9-5.1×10¹¹ virus particles. In some embodiments, the therapeuticsolution comprises 4.95-5.05×10¹¹ virus particles. In some embodiments,the therapeutic solution comprises 4.99-5.01×10¹¹ virus particles. Insome embodiments, the replication defective adenovirus comprises areplication defective adenovirus 5. In some embodiments, the vialfurther comprises an immunogenic component. In some embodiments, theimmunogenic component comprises a cytokine selected from the group of.IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6,IL-9, IL-10, and IL-13. In some embodiments, the immunogenic componentis selected from the group consisting of IL-7, a nucleic acid encodingIL-7, a protein with substantial identity to IL-7, and a nucleic acidencoding a protein with substantial identity to IL-7.

In various aspects, the invention relates to methods of treatment withthe compositions described herein, wherein the treatment comprisesraising an immune response against CEA. In one aspect, the inventionrelates to a method of treatment comprising: (a) selecting a first phaseand a second phase of treatment; (b) during the first phase,administering to a human a total of 3 times, in about 3 week intervals,a first replication defective adenovirus encoding an antigen that iscapable of inducing an immune response directed towards cells expressingCEA antigen in a human; and (c) during the second phase, administeringto said human a total of 3 times, in about 3 month intervals, a secondreplication defective adenovirus encoding an antigen that is capable ofinducing an immune response directed towards cells expressing CEAantigen in a human; wherein the second phase starts about 3 months afterthe end of the first phase. In some embodiments, the first replicationdefective adenovirus encoding an antigen that is capable of inducing animmune response directed towards cells expressing CEA antigen in a humanand the second replication defective adenovirus encoding an antigen thatis capable of inducing an immune response directed towards cellsexpressing CEA antigen in a human are the same. In some embodiments, thefirst replication defective adenovirus comprises a replication defectiveadenovirus 5. In some embodiments, the second replication defectiveadenovirus comprises a replication defective adenovirus 5. In someembodiments, the first phase is at least five weeks. In someembodiments, the second phase is at least 5 months. In some embodiments,the immune response is measured as CEA antigen specific cell-mediatedimmunity (CMI). In some embodiments, the immune response is measured asCEA antigen specific IFN-γ secretion. In some embodiments, the immuneresponse is measured as CEA antigen specific IL-2 secretion. In someembodiments, the immune response against CEA is measured by ELISspotassay. In some embodiments, the CEA antigen specific CMI is greater than300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral blood mononuclearcells (PBMC). In some embodiments, the immune response is measured by Tcell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic CEAexpressing cells from a tumor cell line or from an autologous tumor. Insome embodiments, the first or second replication defective adenovirusinfects dendritic cells in the human and wherein the infected dendriticcells present CEA antigen, thereby inducing the immune response. In someembodiments, the administering steps comprise subcutaneousadministration. In some embodiments, the administering step in b or ccomprises delivering 4.8-5.2×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.9-5.1×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.95-5.05×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.99-5.01×10¹¹ replication defective adenovirusparticles. In some embodiments, the human carries an inverse Ad5neutralizing antibody titer that is of greater than 200 prior to theadministering step. In some embodiments, the human has an inverse Ad5neutralizing antibody titer of greater than 4767. In some embodiments,the human is not concurrently being treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy. In someembodiments, the human has not been treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy prior tothe administering step. In some embodiments, the human does not have anautoimmune disease. In some embodiments, the human does not haveinflammatory bowel disease, systemic lupus erythematosus, ankylosingspondylitis, scleroderma, multiple sclerosis, viral hepatitis, or HIV.In some embodiments, the human is not undergoing cytotoxic chemotherapyconcurrently. In some embodiments, the human is not undergoing radiationtherapy concurrently. In some embodiments, the human has autoimmunerelated thyroid disease or vitiligo. In some embodiments, the human hascolorectal adenocarcinoma, metastatic colorectal cancer, advanced CEAexpressing colorectal cancer, breast cancer, lung cancer, bladdercancer, or pancreas cancer. In some embodiments, the human has threesites of metastatic disease. In some embodiments, the human has receivedchemotherapy prior to the administering step. In some embodiments, priorto the first phase, the human has received at least one medication ofthe group consisting of fluoropyrimidine, irinotecan, oxaliplatin,bevacizunab, Capecitabine, Mitomycin, Regorafenib, cetuxinab, andpanitumumab. In some embodiments, the human concurrently receiveschemotherapy or radiation therapy treatment. In some embodiments, thehuman concurrently receives a therapy comprising the administration ofat least one medication of the group consisting of fluoropyrimidine,irinotecan, oxaliplatin, bevacizunab, Capecitabine, Mitomycin,Regorafenib, cetuxinab, panitumumab, and acetinophen. In someembodiments, the human comprises cells overexpressing CEA. In someembodiments, the cells overexpressing CEA overexpress CEA by at least 10times over the baseline CEA expression in a non-cancer cell. In someembodiments, cells overexpressing CEA comprise cancer cells. In someembodiments, cells overexpressing CEA are not gastrointestinalepithelium cells. In some embodiments, cells overexpressing CEAoverexpress CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, PSG1, PSG2,PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9, or PSG11. In some embodiments,the human expresses a human leukocyte antigen of serotype HLA-A2,HLA-A3, or HLA-A24. In some embodiments, the first or second replicationdefective adenovirus comprises a recombinant nucleic acid vectorcomprising a sequence encoding a modified CEA; wherein the modified CEAcomprises a modification in up to 5 amino acids; and wherein therecombinant nucleic acid vector comprises a replication defectiveadenovirus 5 vector having a deletion in the E2b region. In someembodiments, the first or second replication defective adenoviruscomprises a recombinant nucleic acid vector comprising a sequence with afirst identity value of at least 80% to SEQ ID NO: 3. In someembodiments, the recombinant nucleic acid vector further comprises aregion with a second identity value of at least 80% to a region in SEQID NO: 3, wherein the region is selected from 26048-26177, 26063-26141,1-103, 54-103, 32214-32315, and 32214-32262. In some embodiments, therecombinant nucleic acid vector further comprises a region encoding apeptide with a third identity value of at least 80% to a peptide encodedby a region in SEQ ID NO: 3 between positions 1057 and 3165. In someembodiments, the first or second replication defective adenoviruscomprises a recombinant nucleic acid vector comprising a sequenceencoding a modified CEA; wherein the modified CEA comprises a sequencewith a first identity value of at least 80% to SEQ ID NO: 2; and whereinthe recombinant nucleic acid vector comprises a replication defectiveadenovirus vector.

In a further aspect, the invention relates to a method of treatmentcomprising: (a) selecting a first phase and a second phase of treatment;(b) during the first phase, administering to a human, a total of ntimes, a first replication defective adenovirus encoding an antigen thatis capable of inducing an immune response directed towards cellsexpressing CEA antigen in a human; and (c) during the second phase,administering said human, a total of m times, a second replicationdefective adenovirus encoding an antigen that is capable of inducing animmune response directed towards cells expressing CEA antigen in ahuman. In some embodiments, the first replication defective adenovirusencoding an antigen that is capable of inducing an immune responsedirected towards cells expressing CEA antigen in a human and the secondreplication defective adenovirus encoding an antigen that is capable ofinducing an immune response directed towards cells expressing CEAantigen in a human are the same. In some embodiments, the firstreplication defective adenovirus comprises a replication defectiveadenovirus 5. In some embodiments, the second replication defectiveadenovirus comprises a replication defective adenovirus 5. In someembodiments, n is greater than 1. In some embodiments, n is 3. In someembodiments, m is greater than 1. In some embodiments, m is 3. In someembodiments, the first phase is about six weeks. In some embodiments,the first phase is at least five weeks. In some embodiments, the secondphase is at least 5 months. In some embodiments, the second phase starts3 weeks-16 weeks after first phase ends. In some embodiments, in thefirst phase two administrations of the replication defective adenovirusare at least 18 days apart. In some embodiments, in the first phase twoadministrations of the replication defective adenovirus are about 21days apart. In some embodiments, in the first phase two administrationsof the replication defective adenovirus are at most 24 days apart. Insome embodiments, in the second phase two administrations of thereplication defective adenovirus are at least 10 weeks apart. In someembodiments, in the second phase two administrations of the replicationdefective adenovirus are about 13 weeks apart. In some embodiments, inthe second phase two administrations of the replication defectiveadenovirus are at most 16 weeks apart. In some embodiments, the immuneresponse is measured as CEA antigen specific cell-mediated immunity(CMI). In some embodiments, the immune response is measured as CEAantigen specific IFN-γ secretion. In some embodiments, the immuneresponse is measured as CEA antigen specific IL-2 secretion. In someembodiments, the immune response against CEA is measured by ELISspotassay. In some embodiments, the CEA antigen specific CMI is greater than300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral blood mononuclearcells (PBMC). In some embodiments, the immune response is measured by Tcell lysis of CAP-1 pulsed antigen-presenting cells, allogeneic CEAexpressing cells from a tumor cell line or from an autologous tumor. Insome embodiments, the first or second replication defective adenovirusinfects dendritic cells in the human and wherein the infected dendriticcells present CEA antigen, thereby inducing the immune response. In someembodiments, the administering steps comprise subcutaneousadministration. In some embodiments, the administering step in b or ccomprises delivering 4.8-5.2×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.9-5.1×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.95-5.05×10¹¹ replication defective adenovirusparticles. In some embodiments, the administering step in b or ccomprises delivering 4.99-5.01×10¹¹ replication defective adenovirusparticles. In some embodiments, the human carries an inverse Ad5neutralizing antibody titer that is of greater than 200 prior to theadministering step. In some embodiments, the human has an inverse Ad5neutralizing antibody titer of greater than 4767. In some embodiments,the human is not concurrently being treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy. In someembodiments, the human has not been treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy prior tothe administering step. In some embodiments, the human does not have anautoimmune disease. In some embodiments, the human does not haveinflammatory bowel disease, systemic lupus erythematosus, ankylosingspondylitis, scleroderma, multiple sclerosis, viral hepatitis, or HIV.In some embodiments, the human is not undergoing cytotoxic chemotherapyconcurrently. In some embodiments, the human is not undergoing radiationtherapy concurrently. In some embodiments, the human has autoimmunerelated thyroid disease or vitiligo. In some embodiments, the human hascolorectal adenocarcinoma, metastatic colorectal cancer, advanced CEAexpressing colorectal cancer breast cancer, lung cancer, bladder canceror pancreas cancer. In some embodiments, the human has three sites ofmetastatic disease. In some embodiments, the human has receivedchemotherapy prior to the administering step. In some embodiments, priorto the first phase, the human has received at least one medication ofthe group consisting of fluoropyrimidine, irinotecan, oxaliplatin,bevacizunab, Capecitabine, Mitomycin, Regorafenib, cetuxinab, andpanitumumab. In some embodiments, the human concurrently receiveschemotherapy or radiation therapy treatment. In some embodiments, thehuman concurrently receives a therapy comprising the administration ofat least one medication of the group consisting of fluoropyrimidine,irinotecan, oxaliplatin, bevacizunab, Capecitabine, Mitomycin,Regorafenib, cetuxinab, panitumumab, and acetinophen. In someembodiments, the human comprises cells overexpressing CEA. In someembodiments, the cells overexpressing CEA overexpress CEA more than 20times over the baseline CEA expression in a non-cancer cell. In someembodiments, cells overexpressing CEA comprise cancer cells. In someembodiments, cells overexpressing CEA are not gastrointestinalepithelium cells. In some embodiments, cells overexpressing CEAoverexpress CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, PSG1, PSG2,PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9, or PSG11. In some embodiments,the human expresses a human leukocyte antigen of serotype HLA-A2,HLA-A3, or HLA-A24. In some embodiments, the first or second replicationdefective adenovirus comprises a recombinant nucleic acid vectorcomprising a sequence encoding a modified CEA; wherein the modified CEAcomprises a modification in up to 5 amino acids; and wherein therecombinant nucleic acid vector comprises a replication defectiveadenovirus 5 vector having a deletion in the E2b region. In someembodiments, the first or second replication defective adenoviruscomprises a recombinant nucleic acid vector comprising a sequence with afirst identity value of at least 80% to SEQ ID NO: 3. In someembodiments, the recombinant nucleic acid vector further comprises aregion with a second identity value of at least 80% to a region in SEQID NO: 3, wherein the region is selected from 26048-26177, 26063-26141,1-103, 54-103, 32214-32315, and 32214-32262. In some embodiments, therecombinant nucleic acid vector further comprises a region encoding apeptide with a third identity value of at least 80% to a peptide encodedby a region in SEQ ID NO: 3 between positions 1057 and 3165. In someembodiments, the first or second replication defective adenoviruscomprises a recombinant nucleic acid vector comprising a sequenceencoding a modified CEA; wherein the modified CEA comprises a sequencewith a first identity value of at least 80% to SEQ ID NO: 2; and whereinthe recombinant nucleic acid vector comprises a replication defectiveadenovirus vector.

In another aspect, the invention relates to a method of generating animmune response against CEA in a human, the method comprising: (a)administering to the human the composition as in any of the previousaspects described herein, thereby increasing the immune response to CEAby at least 25 fold. In some embodiments, the immune response ismeasured as CEA antigen specific cell-mediated immunity (CMI). In someembodiments, the immune response is measured as CEA antigen specificIFN-γ secretion. In some embodiments, the immune response is measured asCEA antigen specific IL-2 secretion. In some embodiments, the immuneresponse against CEA is measured by ELISspot assay. In some embodiments,the CEA antigen specific CMI is greater than 300 IFN-γ spot formingcells (SFC) per 10⁶ peripheral blood mononuclear cells (PBMC). In someembodiments, the immune response is measured by T cell lysis of CAP-1pulsed antigen-presenting cells, allogeneic CEA expressing cells from atumor cell line or from an autologous tumor. In some embodiments, therecombinant nucleic acid vector infects dendritic cells in the human andwherein the infected dendritic cells present CEA antigen, therebyinducing the immune response. In some embodiments, the administeringstep comprises subcutaneous administration. In some embodiments, theadministering step is repeated at least once. In some embodiments, theadministering step is repeated after about 3 weeks following a previousadministering step. In some embodiments, the administering step isrepeated after about 3 months following a previous administering step.In some embodiments, the administering step is repeated twice. In someembodiments, the composition comprises 4.8-5.2×10¹¹ virus particlescomprising the recombinant nucleic acid vector. In some embodiments, thecomposition comprises 4.9-5.1×10¹¹ virus particles comprising therecombinant nucleic acid vector. In some embodiments, the compositioncomprises 4.95-5.05×10¹¹ virus particles comprising the recombinantnucleic acid vector. In some embodiments, the composition comprises4.99-5.01×10¹¹ virus particles comprising the recombinant nucleic acidvector. In some embodiments, the human carries an Ad5 neutralizingantibody titer that is of greater than 200 prior to the administeringstep. In some embodiments, the human has an inverse Ad5 neutralizingantibody titer of greater than 4767. In some embodiments, the human isnot concurrently being treated by any one of steroids, corticosteroids,immunosuppressive agents, and immunotherapy. In some embodiments, thehuman has not been treated by any one of steroids, corticosteroids,immunosuppressive agents, and immunotherapy prior to the administeringstep. In some embodiments, the human does not have an autoimmunedisease. In some embodiments, the human does not have inflammatory boweldisease, systemic lupus erythematosus, ankylosing spondylitis,scleroderma, multiple sclerosis, viral hepatitis, or HIV. In someembodiments, the human is not undergoing cytotoxic chemotherapyconcurrently. In some embodiments, the human is not undergoing radiationtherapy concurrently. In some embodiments, the human has autoimmunerelated thyroid disease or vitiligo. In some embodiments, the human hascolorectal adenocarcinoma, metastatic colorectal cancer, advanced CEAexpressing colorectal cancer breast cancer, lung cancer, bladder cancer,or pancreas cancer. In some embodiments, the human has three sites ofmetastatic disease. In some embodiments, the human has receivedchemotherapy prior to the administering step. In some embodiments, priorto the administering step, the human has received at least onemedication of the group consisting of fluoropyrimidine, irinotecan,oxaliplatin, bevacizunab, Capecitabine, Mitomycin, Regorafenib,cetuxinab, and panitumumab. In some embodiments, the human concurrentlyreceives chemotherapy or radiation therapy treatment. In someembodiments, the human concurrently receives a therapy comprising theadministration of at least one medication of the group consisting offluoropyrimidine, irinotecan, oxaliplatin, bevacizunab, Capecitabine,Mitomycin, Regorafenib, cetuxinab, panitumumab, and acetinophen. In someembodiments, the human comprises cells overexpressing CEA. In someembodiments, the cells overexpressing CEA overexpress CEA more than 10times over the baseline CEA expression in a non-cancer cell. In someembodiments, cells overexpressing CEA comprise cancer cells. In someembodiments, cells overexpressing CEA are not gastrointestinalepithelium cells. In some embodiments, cells overexpressing CEAoverexpress CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, PSG1, PSG2,PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9, or PSG11. In some embodiments,the human expresses a human leukocyte antigen of serotype HLA-A2,HLA-A3, or HLA-A24.

In yet another aspect, the invention relates to a method of selecting ahuman for administration of the composition as in any of the previousaspects described herein, comprising; (a) determining a HLA subtype ofthe human; and (b) administering the composition to the human, if theHLA subtype is determined to be one of a preselected subgroup of HLAsubtypes. In some embodiments, the preselected subgroup of HLA subtypescomprises one or more of HLA-A2, HLA-A3, or HLA-A24. In someembodiments, the method induces a CEA-specific immune response that ismeasured as CEA antigen specific cell-mediated immunity (CMI). In someembodiments, the method induces a CEA-specific immune response that ismeasured as CEA antigen specific IFN-γ secretion. In some embodiments,the method induces a CEA-specific immune response that is measured asCEA antigen specific IL-2 secretion. In some embodiments, the methodinduces a CEA-specific immune response against CEA that is measured byELISspot assay. In some embodiments, the CEA antigen specific CMI isgreater than 300 IFN-γ spot forming cells (SFC) per 10⁶ peripheral bloodmononuclear cells (PBMC). In some embodiments, the method induces aCEA-specific immune response that is measured by T cell lysis of CAP-1pulsed antigen-presenting cells, allogeneic CEA expressing cells from atumor cell line or from an autologous tumor. In some embodiments, therecombinant nucleic acid vector infects dendritic cells in the human andwherein the infected dendritic cells present CEA antigen, therebyinducing an immune response. In some embodiments, the administering stepcomprises subcutaneous administration. In some embodiments, theadministering step is repeated at least once. In some embodiments, theadministering step is repeated after about 3 weeks following a previousadministering step. In some embodiments, the administering step isrepeated after about 3 months following a previous administering step.In some embodiments, the administering step is repeated twice. In someembodiments, the composition comprises 4.8-5.2×10¹¹ virus particlescomprising the recombinant nucleic acid vector. In some embodiments, thecomposition comprises 4.9-5.1×10¹¹ virus particles comprising therecombinant nucleic acid vector. In some embodiments, the compositioncomprises 4.95-5.05×10¹¹ virus particles comprising the recombinantnucleic acid vector. In some embodiments, the composition comprises4.99-5.01×10¹¹ virus particles comprising the recombinant nucleic acidvector. In some embodiments, the human carries an inverse Ad5neutralizing antibody titer that is of greater than 200 prior to theadministering step. In some embodiments, the human has an inverse Ad5neutralizing antibody titer of greater than 4767. In some embodiments,the human is not concurrently being treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy. In someembodiments, the human has not been treated by any one of steroids,corticosteroids, immunosuppressive agents, and immunotherapy prior tothe administering step. In some embodiments, the human does not have anautoimmune disease. In some embodiments, the human does not haveinflammatory bowel disease, systemic lupus erythematosus, ankylosingspondylitis, scleroderma, multiple sclerosis, viral hepatitis, or HIV.In some embodiments, the human is not undergoing cytotoxic chemotherapyconcurrently. In some embodiments, the human is not undergoing radiationtherapy concurrently. In some embodiments, the human has autoimmunerelated thyroid disease or vitiligo. In some embodiments, the human hascolorectal adenocarcinoma, metastatic colorectal cancer, advanced CEAexpressing colorectal cancer breast cancer, lung cancer, bladder cancer,or pancreas cancer. In some embodiments, the human has three sites ofmetastatic disease. In some embodiments, the human has receivedchemotherapy prior to the administering step. In some embodiments, priorto the administering step, the human has received at least onemedication of the group consisting of fluoropyrimidine, irinotecan,oxaliplatin, bevacizunab, Capecitabine, Mitomycin, Regorafenib,cetuxinab, and panitumumab. In some embodiments, the human concurrentlyreceives chemotherapy or radiation therapy treatment. In someembodiments, the human concurrently receives a therapy comprising theadministration of at least one medication of the group consisting offluoropyrimidine, irinotecan, oxaliplatin, bevacizunab, Capecitabine,Mitomycin, Regorafenib, cetuxinab, panitumumab, and acetinophen. In someembodiments, the human comprises cells overexpressing CEA. In someembodiments, the cells overexpressing CEA overexpress CEA more than 10times over the baseline CEA expression in a non-cancer cell. In someembodiments, cells overexpressing CEA comprise cancer cells. In someembodiments, cells overexpressing CEA are not gastrointestinalepithelium cells. In some embodiments, cells overexpressing CEAoverexpress CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21, PSG1, PSG2,PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9, or PSG11.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a bar graph showing antibody levels from mice immunized withAd5Null. Mice were immunized three times with Ad5Null viral particles at14 day intervals. Note the presence of increasing anti-Ad antibodylevels after each immunization.

FIG. 2 is a bar graph showing neutralizing antibody levels from miceimmunized with Ad5Null. Mice were immunized three times with Ad5Nullviral particles at 14 day intervals. Note the presence of increasingneutralizing antibodies after each immunization. Optical densityreadings indicate the presence of viable target cells.

FIG. 3 is a bar graph showing the induction of NAb in C57BI/6 mice afterinjections with Ad5-Null vector platform (VP). Note the increasinglevels of NAb induced in mice after repeated injections with Adparticles. Values represent mean±SEM.

FIG. 4A is a bar graph showing INF-.γ. secreting splenocytes from Ad5immune mice immunized with Ad5 [E1-]-CEA or Ad5 [E1, E2b-]-CEA. Note thesignificantly elevated response in splenocytes from the Ad5 [E1-,E2b]-CEA immunized group. Values represent mean±SEM.

FIG. 4B is a bar graph showing IL-2 secreting splenocytes from Ad5immune mice immunized with Ad5 [E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Notethe significantly elevated response in splenocytes from the Ad5 [E1-,E2b]-CEA immunized group. Values represent mean±SEM.

FIG. 5 is a bar graph showing serum AST levels in control mice and micevaccinated with 10¹⁰ viral particles of Ad5 [E1-]-CEA or Ad5 [E1-,E2b-]-CEA. Values represent mean±SEM.

FIG. 6 is a line graph showing tumor volume. Ad5 immune C57BI/6 micewere injected with MC38 CEA expressing tumor cells and subsequentlytreated (Vac) with Ad5 [E1-, E2b-]-CEA vaccine as described. Note thesignificant reduction in tumor size by days 19-21 as compared tountreated control tumor bearing mice. Tumor measurements were taken andvolumes were determined. Statistical analysis was performed using theBonferroni post-tests analysis with PRISM software. Values representmean±SEM.

FIG. 7 is a graph showing tumor weights from treated and untreated Ad5immune MC38 tumor bearing mice. Note the significant (p=0.0124)reduction in tumor weights from the mice treated with Ad5 [E1-,E2b-]-CEA. Values represent mean±SEM.

FIG. 8 is a representative immunoblot demonstrating Gag production byA-549 cells infected with Ad5 [E1-, E2b-]-gag. A-549 whole cell lysatewas infected at a MOI of 200 of Ad5 [E1-, E2b-]-gag or Ad5-null for 44h. The blot was stained with a mouse monoclonal antibody against Gag.Lane 1, Magic Mark XP Western Standard (Invitrogen, CA), Lanes 2 and 3,Ad5 [E1-, E2b-]-gag, Lane 4, Ad5-null (empty). The upper band (55 kDa)comprises the gag precursor and the lower band (41 kDa) comprises thep17/p24 gag complex.

FIG. 9 demonstrates the effect of multiple immunizations on inducing agreater CMI (IFN-γ ELISpot) response. Ad5 Naïve BALB/C mice (n=5/group)were immunized once or three times at fourteen day intervals with 10¹⁰VP of Ad5 [E1-]-null, Ad5 [E1-, E2b-]-null, Ad5 [E1-]-gag, Ad5 [E1-,E2b-]-gag, or injection buffer alone (control). Fourteen days after thefinal immunization splenocytes were assessed for IFN-γ secretingsplenocytes by ELISpot analysis. For positive controls, splenocytes wereexposed to Concanavalin A (Con A) (data not shown). The error barsdepict the SEM

FIG. 10 shows the results of ELISpot INF-γ (A) and IL-2 (B) analysis ofPBMC from Cynomolgus Macaques (N=3) pre-immunized against Wild Type Ad5.When Ad5 neutralizing antibody titers reached 1:50 or greater they wereimmunized intradermally three times at 30 day intervals with Ad5-[E1-,E2b-]-gag at a dose of 10¹⁰ VP. The first immunization (Wild Type Ad5)was on day 1 and 124 days later (32 days after last vaccination) the NAbtiters were equal to or greater than 1:1000. Note the presence ofsignificantly elevated values (P<0.05) in the Dec. 17, 2007 samples. Forpositive controls, splenocytes were exposed to Concanavalin A (Con A)(data not shown). Values represent mean±SEM.

FIG. 11 ELISpot interferon-γ secreting SPC from mice vaccinated withrecombinant Ad5 CEA expression vectors. Graph shows lack of reduction inSPC from mice vaccinated with Ad5 [[E1-, E2b-]-CEA as compared with thereductions in SPC from Ad5 [E1-]-CEA vaccinated mice.

FIG. 12 demonstrates Kaplan-Meier survival plots of patients treatedwith Ad5 [E1-, E2b-]-CEA(6D). Patients treated with Ad5 [E1-,E2b-]-CEA(6D) were followed for survival. Panel A represents 7 patientsin cohorts 1 and 2 that were followed for survival. There were 5 eventsin this group. Panel B represents 21 patients in cohort 3 and Ph II thatwere followed for survival. There were 11 events in this group. Panel Crepresents 6 patients in cohort 5 that were followed for survival. Therewere 3 events in this group. Panel D represents all 34 that werefollowed for survival. There were 19 events in this group.

FIG. 13 demonstrates Kaplan-Meier survival plots of patients treatedwith Ad5 [E1-, E2b-]-CEA(6D). Patients treated three times with Ad5[E1-, E2b-]-CEA(6D) were followed for survival. Panel A represents 28patients that were followed for survival. There were 11 events in thisgroup. Panel B represents 27 patients that were followed for survival.There were 11 events in this group.

FIG. 14 demonstrates CEA-directed CMI responses in treated patients. CMI(IFN-γ secretion) was assessed at baseline (Pre) and afteradministrations of Ad5 [E1-, E2b-]-CEA(6D) (Post). The highest CMIresponses (regardless of time point) observed in the patients aftertreatment revealed a dose response. The highest CMI levels occurred inpatients that received the highest dose of 5×10″ VP (Cohort 5). The CMIresponses for Cohort 3/Phase II and Cohort 5 were significantly elevated(P=0.0002 and P=0.0317, respectively; Mann-Whitney test) as compared totheir baseline (Pre) values. Specificity of the responses wasdemonstrated by the lack of reactivity with the irrelevant antigensβ-galactosidase and HIV-gag (data not shown). For positive controls,PBMCs were exposed to concanavalin A (data not shown). Values=Mean±SEMfor each Cohort.

FIG. 15 demonstrates CEA directed CMI responses in treated patients. CMI(IFN-γ secretion) was assessed at baseline (week 0) and 3 weeks afterthe last immunotherapy (week 9) for patients in all 4-dose cohorts. Adose response was observed and the highest CMI level occurred inpatients that received the highest dose. The CMI response with thehighest dose was significantly elevated (P<0.02; Mann-Whitney test).Specificity of the responses was demonstrated by the lack of reactivitywith the irrelevant antigens β-galactosidase and HIV-gag (data notshown). For positive controls, PBMCs were exposed to concanavalin A(data not shown). Horizontal line and error bar indicate the mean±SEM.

FIG. 16 demonstrates Ad5 immune responses in patients receivingimmunizations with Ad5 [E1-, E2b-]-CEA(6D) vaccine. Ad5 NAb titers (A)and CMI responses (B) to Ad5 were determined in patients at baseline(week 0) and 3 weeks (week 9) after the third immunization. The numberof IFN-γ secreting PBMCs from patients that were specific for Ad5 wasdetermined by ELISpot. Both the Ad5 NAb titers and Ad5 CMI responseswere significantly elevated at week 9 (P<0.0001 and P<0.01,respectively; Mann-Whitney test). Values=Mean±SEM.

FIG. 17 demonstrates CEA-specific immunity in patients receivingimmunizations with Ad5 [E1-, E2b-]-CEA(6D) vaccine and comparisons withAd5 immunity. (A) The mean CEA specific immune responses in patients(n=19) who received 1×10¹¹ VP of Ad5 [E1-, E2b-]-CEA(6D) as measured byIFN-γ secretion of PBMC in patients with none to low pre-existing Ad5immunity (NAb<200, white bars) as compared to the CEA specific immuneresponse of patients with high pre-existing Ad5 immunity (NAb titer≧200,black bars) prior to the initiation of treatment with Ad5 [E1-,E2b-]-CEA(6D). There was no significant difference between the twogroups at any time point tested (P>0.4, Mann-Whitney test)(Values=Mean±SEM). (B) Correlation between pre-existing Ad5 NAb activityand highest levels of induced CEA CMI responses. (C) Correlation betweenvector induced Ad5 NAb activity and CEA CMI responses. The r² values inB and C revealed no correlation between pre-existing or vector inducedAd5 NAb activity and CEA CMI ELISpot responses.

DETAILED DESCRIPTION

The present invention relates to methods and adenovirus vectors forgenerating immune responses against target antigens, in particular,those related to cancer cells. In various aspects of the invention,compositions and methods described herein relate to generating an immuneresponse against cells expressing and/or presenting a target antigen,such as colorectal embryonic antigen (CEA). In some embodiments, amodified form of CEA is used in a vaccine directed to raising an immuneresponse against CEA or cells expressing and/or presenting CEA. Inparticular, the present invention provides an improved adenovirus(Ad)-based vaccine such that multiple vaccinations against one or moreantigenic target entity can be achieved. In some embodiments, theimproved adenovirus (Ad)-based vaccine comprises a replication defectiveadenovirus carrying a target antigen, a fragment, a variant or a variantfragment thereof, such as Ad5 [E1-,E2b-]-CEA(6D). Variants and/orfragments of target antigens, for example CEA, can be selected based ona variety of factors, including immunogenic potential. Accordingly, amutant CEA, CEA(6D) is utilized in various embodiments of the inventionfor its increased capability to raise an immune response relative to thewild type form. Importantly, vaccination can be performed in thepresence of preexisting immunity to the Ad and/or administered tosubjects previously immunized multiple times with the adenovirus vectorof the present invention or other adenovirus vectors. The adenovirusvectors of the invention can be administered to subjects multiple timesto induce an immune response against an antigen of interest, for exampleCEA, including but not limited to, the production of antibodies andcell-mediated immune responses against one or more target antigens.

The following passages describe different aspects of the invention ingreater detail. Each aspect of the invention may be combined with anyother aspect or aspects of the invention unless clearly indicated to thecontrary. In particular, any feature indicated as being preferred oradvantageous may be combined with any other feature of featuresindicated as being preferred or advantageous.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, unless otherwise indicated, the article “a” means one ormore unless explicitly otherwise provided for.

As used herein, unless otherwise indicated, terms such as “contain,”“containing,” “include,” “including,” and the like mean “comprising.”

As used herein, unless otherwise indicated, the term “or” can beconjunctive or disjunctive.

As used herein, unless otherwise indicated, any embodiment can becombined with any other embodiment.

As used herein, unless otherwise indicated, some inventive embodimentsherein contemplate numerical ranges. A variety of aspects of thisinvention can be presented in a range format. It should be understoodthat the description in range format is merely for convenience andbrevity and should not be construed as an inflexible limitation on thescope of the invention. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible subrangesas well as individual numerical values within that range as ifexplicitly written out. For example, description of a range such as from1 to 6 should be considered to have specifically disclosed subrangessuch as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,from 3 to 6 etc., as well as individual numbers within that range, forexample, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth ofthe range. When ranges are present, the ranges include the rangeendpoints.

The term “adenovirus” or “Ad” refers to a group of non-enveloped DNAviruses from the family Adenoviridae. In addition to human hosts, theseviruses can be found in, but are not limited to, avian, bovine, porcineand canine species. The present invention contemplates the use of anyadenovirus from any of the four genera of the family Adenoviridae (e.g.,Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus) as thebasis of an E2b deleted virus vector, or vector containing otherdeletions as described herein. In addition, several serotypes are foundin each species. Ad also pertains to genetic derivatives of any of theseviral serotypes, including but not limited to, genetic mutation,deletion or transposition of homologous or heterologous DNA sequences.

A “helper adenovirus” or “helper virus” refers to an Ad that can supplyviral functions that a particular host cell cannot (the host may provideAd gene products such as E1 proteins). This virus is used to supply, intrans, functions (e.g., proteins) that are lacking in a second virus, orhelper dependent virus (e.g., a gutted or gutless virus, or a virusdeleted for a particular region such as E2b or other region as describedherein); the first replication-incompetent virus is said to “help” thesecond, helper dependent virus thereby permitting the production of thesecond viral genome in a cell.

The term “Adenovirus5 null (Ad5null)”, as used herein, refers to anon-replicating Ad that does not contain any heterologous nucleic acidsequences for expression.

The term “First Generation adenovirus”, as used herein, refers to an Adthat has the early region 1 (E1) deleted. In additional cases, thenonessential early region 3 (E3) may also be deleted.

The term “gutted” or “gutless”, as used herein, refers to an adenovirusvector that has been deleted of all viral coding regions.

The term “transfection” as used herein refers to the introduction offoreign nucleic acid into eukaryotic cells. Transfection may beaccomplished by a variety of means known to the art including calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign nucleic acid, DNA or RNA, intothe genome of the transfected cell. The term “stable transfectant”refers to a cell which has stably integrated foreign DNA into thegenomic DNA.

The term “reporter gene” indicates a nucleotide sequence that encodes areporter molecule (including an enzyme). A “reporter molecule” isdetectable in any of a variety of detection systems, including, but notlimited to enzyme-based detection assays (e.g., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. In one embodiment, the present inventioncontemplates the E. coli β-galactosidase gene (available from PharmaciaBiotech, Pistacataway, N.J.), green fluorescent protein (GFP)(commercially available from Clontech, Palo Alto, Calif.), the humanplacental alkaline phosphatase gene, the chloramphenicolacetyltransferase (CAT) gene; other reporter genes are known to the artand may be employed.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The nucleic acid sequence thus codesfor the amino acid sequence.

The term “heterologous nucleic acid sequence”, as used herein, refers toa nucleotide sequence that is ligated to, or is manipulated to becomeligated to, a nucleic acid sequence to which it is not ligated innature, or to which it is ligated at a different location in nature.Heterologous nucleic acid may include a nucleotide sequence that isnaturally found in the cell into which it is introduced or theheterologous nucleic acid may contain some modification relative to thenaturally occurring sequence.

The term “transgene” refers to any gene coding region, either natural orheterologous nucleic acid sequences or fused homologous or heterologousnucleic acid sequences, introduced into the cells or genome of a testsubject. In the current invention, transgenes are carried on any viralvector that is used to introduce the transgenes to the cells of thesubject.

The term “Second Generation Adenovirus”, as used herein, refers to an Adthat has all or parts of the E1, E2, E3, and, in certain embodiments, E4DNA gene sequences deleted (removed) from the virus.

The term “subject”, as used herein, refers to any animal, e.g., a mammalor marsupial. Subjects of the present invention include but are notlimited to humans, non-human primates (e.g., rhesus or other types ofmacaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowl ofany kind.

In particular embodiments, the present invention relates to areplication defective adenovirus vector of serotype 5 comprising asequence encoding an immunogenic polypeptide. The immunogenicpolypeptide may be a mutant CEA or a fragment thereof. In someembodiments, the immunogenic polypeptide comprises a mutant CEA with anAsn->Asp substitution at position 610. In some embodiments, thereplication defective adenovirus vector comprises a sequence encoding apolypeptide with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%,99.9% identity to the immunogenic polypeptide. In some embodiments, thesequence encoding the immunogenic polypeptide comprises the followingsequence identified by SEQ. ID. NO: 1:

(SEQ. ID. NO.: 1) ATGGAGTCTCCCTCGGCCCCTCCCCACAGATGGTGCATCCCCTGGCAGAGGCTCCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCAAGCTCACTATTGAATCCACGCCGTTCAATGTCGCAGAGGGGAAGGAGGTGCTTCTACTTGTCCACAATCTGCCCCAGCATCTTTTTGGCTACAGCTGGTACAAAGGTGAAAGAGTGGATGGCAACCGTCAAATTATAGGATATGTAATAGGAACTCAACAAGCTACCCCAGGGCCCGCATACAGTGGTCGAGAGATAATATACCCCAATGCATCCCTGCTGATCCAGAACATCATCCAGAATGACACAGGATTCTACACCCTACACGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGCCAGTTCCGGGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACGCAACCTACCTGTGGTGGGTAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACACAGCAAGCTACAAATGTGAAACCCAGAACCCAGTGAGTGCCAGGCGCAGTGATTCAGTCATCCTGAATGTCCTCTATGGCCCGGATGCCCCCACCATTTCCCCTCTAAACACATCTTACAGATCAGGGGAAAATCTGAACCTCTCCTGCCACGCAGCCTCTAACCCACCTGCACAGTACTCTTGGTTTGTCAATGGGACTTTCCAGCAATCCACCCAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGTGGATCCTATACGTGCCAAGCCCATAACTCAGACACTGGCCTCAATAGGACCACAGTCACGACGATCACAGTCTATGCAGAGCCACCCAAACCCTTCATCACCAGCAACAACTCCAACCCCGTGGAGGATGAGGATGCTGTAGCCTTAACCTGTGAACCTGAGATTCAGAACACAACCTACCTGTGGTGGGTAAATAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGACAACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGATGTAGGACCCTATGAGTGTGGAATCCAGAACGAATTAAGTGTTGACCACAGCGACCCAGTCATCCTGAATGTCCTCTATGGCCCAGACGACCCCACCATTTCCCCCTCATACACCTATTACCGTCCAGGGGTGAACCTCAGCCTCTCCTGCCATGCAGCCTCTAACCCACCTGCACAGTATTCTTGGCTGATTGATGGGAACATCCAGCAACACACACAAGAGCTCTTTATCTCCAACATCACTGAGAAGAACAGCGGACTCTATACCTGCCAGGCCAATAACTCAGCCAGTGGCCACAGCAGGACTACAGTCAAGACAATCACAGTCTCTGCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGGCTCAGAACACAACCTACCTGTGGTGGGTAAATGGTCAGAGCCTCCCAGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACGCAAGAGCCTATGTATGTGGAATCCAGAACTCAGTGAGTGCAAACCGCAGTGACCCAGTCACCCTGGATGTCCTCTATGGGCCGGACACCCCCATCATTTCCCCCCCAGACTCGTCTTACCTTTCGGGAGCGAACCTCAACCTCTCCTGCCACTCGGCCTCTAACCCATCCCCGCAGTATTCTTGGCGTATCAATGGGATACCGCAGCAACACACACAAGTTCTCTTTATCGCCAAAATCACGCCAAATAATAACGGGACCTATGCCTGTTTTGTCTCTAACTTGGCTACTGGCCGCAATAATTCCATAGTCAAGAGCATCACAGTCTCTGCATCTGGAACTTCTCCTGGTCTCTCAGCTGGGGCCACTGTCGGCATCATGATTGGAGTGCTGGTTGGGGTTGCT CTGATATAG,.

In some embodiments, the sequence encoding the immunogenic polypeptidecomprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%,99.5%, 99.9% identity to SEQ. ID. NO: 1 or a sequence generated fromSEQ. ID. NO: 1 by alternative codon replacements. In some embodiments,the immunogenic polypeptide encoded by the adenovirus vectors describedherein comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or more point mutations, such assingle amino acid substitutions or deletions, as compared to a wild-typehuman CEA sequence.

In some embodiments, the immunogenic polypeptide comprises a sequencefrom SEQ. ID. NO.:2 or a modified version, e.g. comprising up to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, or more point mutations, such as single amino acid substitutionsor deletions, of SEQ. ID. NO.:2:

SEQ. ID. NO.: 2 ATGGAGTCTCCCTCGGCCCCTCCCCACAGATGGTGCATCCCCTGGCAGAGGCTCCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCAAGCTCACTATTGAATCCACGCCGTTCAATGTCGCAGAGGGGAAGGAGGTGCTTCTACTTGTCCACAATCTGCCCCAGCATCTTTTTGGCTACAGCTGGTACAAAGGTGAAAGAGTGGATGGCAACCGTCAAATTATAGGATATGTAATAGGAACTCAACAAGCTACCCCAGGGCCCGCATACAGTGGTCGAGAGATAATATACCCCAATGCATCCCTGCTGATCCAGAACATCATCCAGAATGACACAGGATTCTACACCCTACACGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGCCAGTTCCGGGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACGCAACCTACCTGTGGTGGGTAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACACAGCAAGCTACAAATGTGAAACCCAGAACCCAGTGAGTGCCAGGCGCAGTGATTCAGTCATCCTGAATGTCCTCTATGGCCCGGATGCCCCCACCATTTCCCCTCTAAACACATCTTACAGATCAGGGGAAAATCTGAACCTCTCCTGCCACGCAGCCTCTAACCCACCTGCACAGTACTCTTGGTTTGTCAATGGGACTTTCCAGCAATCCACCCAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGTGGATCCTATACGTGCCAAGCCCATAACTCAGACACTGGCCTCAATAGGACCACAGTCACGACGATCACAGTCTATGCAGAGCCACCCAAACCCTTCATCACCAGCAACAACTCCAACCCCGTGGAGGATGAGGATGCTGTAGCCTTAACCTGTGAACCTGAGATTCAGAACACAACCTACCTGTGGTGGGTAAATAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGACAACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGATGTAGGACCCTATGAGTGTGGAATCCAGAACGAATTAAGTGTTGACCACAGCGACCCAGTCATCCTGAATGTCCTCTATGGCCCAGACGACCCCACCATTTCCCCCTCATACACCTATTACCGTCCAGGGGTGAACCTCAGCCTCTCCTGCCATGCAGCCTCTAACCCACCTGCACAGTATTCTTGGCTGATTGATGGGAACATCCAGCAACACACACAAGAGCTCTTTATCTCCAACATCACTGAGAAGAACAGCGGACTCTATACCTGCCAGGCCAATAACTCAGCCAGTGGCCACAGCAGGACTACAGTCAAGACAATCACAGTCTCTGCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGGCTCAGAACACAACCTACCTGTGGTGGGTAAATGGTCAGAGCCTCCCAGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACGCAAGAGCCTATGTATGTGGAATCCAGAACTCAGTGAGTGCAAACCGCAGTGACCCAGTCACCCTGGATGTCCTCTATGGGCCGGACACCCCCATCATTTCCCCCCCAGACTCGTCTTACCTTTCGGGAGCGAACCTCAACCTCTCCTGCCACTCGGCCTCTAACCCATCCCCGCAGTATTCTTGGCGTATCAATGGGATACCGCAGCAACACACACAAGTTCTCTTTATCGCCAAAATCACGCCAAATAATAACGGGACCTATGCCTGTTTTGTCTCTAACTTGGCTACTGGCCGCAATAATTCCATAGTCAAGAGCATCACAGTCTCTGCATCTGGAACTTCTCCTGGTCTCTCAGCTGGGGCCACTGTCGGCATCATGATTGGAGTGCTGGTTGGGGTTGCT CTGATATAG,.

The compositions and methods of the invention, in some embodiments,relate to an adenovirus-derived vector comprising the following sequenceidentified by SEQ. ID. NO: 3:

(SEQ. ID. NO: 3) CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATACTGTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCTGGTACCGTCGACGCGGCCGCTCGAGCCTAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGCTTAAAGGTACCCAGAGCAGACAGCCGCCACCATGGAGTCTCCCTCGGCCCCTCCCCACAGATGGTGCATCCCCTGGCAGAGGCTCCTGCTCACAGCCTCACTTCTAACCTTCTGGAACCCGCCCACCACTGCCAAGCTCACTATTGAATCCACGCCGTTCAATGTCGCAGAGGGGAAGGAGGTGCTTCTACTTGTCCACAATCTGCCCCAGCATCTTTTTGGCTACAGCTGGTACAAAGGTGAAAGAGTGGATGGCAACCGTCAAATTATAGGATATGTAATAGGAACTCAACAAGCTACCCCAGGGCCCGCATACAGTGGTCGAGAGATAATATACCCCAATGCATCCCTGCTGATCCAGAACATCATCCAGAATGACACAGGATTCTACACCCTACACGTCATAAAGTCAGATCTTGTGAATGAAGAAGCAACTGGCCAGTTCCGGGTATACCCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGACTCAGGACGCAACCTACCTGTGGTGGGTAAACAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACACAGCAAGCTACAAATGTGAAACCCAGAACCCAGTGAGTGCCAGGCGCAGTGATTCAGTCATCCTGAATGTCCTCTATGGCCCGGATGCCCCCACCATTTCCCCTCTAAACACATCTTACAGATCAGGGGAAAATCTGAACCTCTCCTGCCACGCAGCCTCTAACCCACCTGCACAGTACTCTTGGTTTGTCAATGGGACTTTCCAGCAATCCACCCAAGAGCTCTTTATCCCCAACATCACTGTGAATAATAGTGGATCCTATACGTGCCAAGCCCATAACTCAGACACTGGCCTCAATAGGACCACAGTCACGACGATCACAGTCTATGCAGAGCCACCCAAACCCTTCATCACCAGCAACAACTCCAACCCCGTGGAGGATGAGGATGCTGTAGCCTTAACCTGTGAACCTGAGATTCAGAACACAACCTACCTGTGGTGGGTAAATAATCAGAGCCTCCCGGTCAGTCCCAGGCTGCAGCTGTCCAATGACAACAGGACCCTCACTCTACTCAGTGTCACAAGGAATGATGTAGGACCCTATGAGTGTGGAATCCAGAACGAATTAAGTGTTGACCACAGCGACCCAGTCATCCTGAATGTCCTCTATGGCCCAGACGACCCCACCATTTCCCCCTCATACACCTATTACCGTCCAGGGGTGAACCTCAGCCTCTCCTGCCATGCAGCCTCTAACCCACCTGCACAGTATTCTTGGCTGATTGATGGGAACATCCAGCAACACACACAAGAGCTCTTTATCTCCAACATCACTGAGAAGAACAGCGGACTCTATACCTGCCAGGCCAATAACTCAGCCAGTGGCCACAGCAGGACTACAGTCAAGACAATCACAGTCTCTGCGGAGCTGCCCAAGCCCTCCATCTCCAGCAACAACTCCAAACCCGTGGAGGACAAGGATGCTGTGGCCTTCACCTGTGAACCTGAGGCTCAGAACACAACCTACCTGTGGTGGGTAAATGGTCAGAGCCTCCCAGTCAGTCCCAGGCTGCAGCTGTCCAATGGCAACAGGACCCTCACTCTATTCAATGTCACAAGAAATGACGCAAGAGCCTATGTATGTGGAATCCAGAACTCAGTGAGTGCAAACCGCAGTGACCCAGTCACCCTGGATGTCCTCTATGGGCCGGACACCCCCATCATTTCCCCCCCAGACTCGTCTTACCTTTCGGGAGCGGACCTCAACCTCTCCTGCCACTCGGCCTCTAACCCATCCCCGCAGTATTCTTGGCGTATCAATGGGATACCGCAGCAACACACACAAGTTCTCTTTATCGCCAAAATCACGCCAAATAATAACGGGACCTATGCCTGTTTTGTCTCTAACTTGGCTACTGGCCGCAATAATTCCATAGTCAAGAGCATCACAGTCTCTGCATCTGGAACTTCTCCTGGTCTCTCAGCTGGGGCCACTGTCGGCATCATGATTGGAGTGCTGGTTGGGGTTGCTCTGATATAGCAGCCCTGGTGTAGTTTCTTCATTTCAGGAAGACTGACAGTTGTTTTGCTTCTTCCTTAAAGCATTTGCAACAGCTACAGTCTAAAATTGCTTCTTTACCAAGGATATTTACAGAAAAGACTCTGACCAGAGATCGAGACCATCCTCTAGATAAGATATCCGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAACGCGGATCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGCTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTCCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTTGGGCGCGAGAAATACCGATTCCGGGGAGTAGGCATCCGCGCCGCAGGCCCCGCAGACGGTCTCGCATTCCACGAGCCAGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATGAGCCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGTGTCCCCGTATACAGACTTGAGAGGCCTGTCCTCGAGCGGTGTTCCGCGGTCCTCCTCGTATAGAAACTCGGACCACTCTGAGACAAAGGCTCGCGTCCAGGCCAGCACGAAGGAGGCTAAGTGGGAGGGGTAGCGGTCGTTGTCCACTAGGGGGTCCACTCGCTCCAGGGTGTGAAGACACATGTCGCCCTCTTCGGCATCAAGGAAGGTGATTGGTTTGTAGGTGTAGGCCACGTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGGTGAGTACTCCCTCTGAAAAGCGGGCATGACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCATCCATCTGGTCAGAAAAGACAATCTTTTTGTTGTCAAGCTTGGTGGCAAACGACCCGTAGAGGGCGTTGGACAGCAACTTGGCGATGGAGCGCAGGGTTTGGTTTTTGTCGCGATCGGCGCGCTCCTTGGCCGCGATGTTTAGCTGCACGTATTCGCGCGCAACGCACCGCCATTCGGGAAAGACGGTGGTGCGCTCGTCGGGCACCAGGTGCACGCGCCAACCGCGGTTGTGCAGGGTGACAAGGTCAACGCTGGTGGCTACCTCTCCGCGTAGGCGCTCGTTGGTCCAGCAGAGGCGGCCGCCCTTGCGCGAGCAGAATGGCGGTAGGGGGTCTAGCTGCGTCTCGTCCGGGGGGTCTGCGTCCACGGTAAAGACCCCGGGCAGCAGGCGCGCGTCGAAGTAGTCTATCTTGCATCCTTGCAAGTCTAGCGCCTGCTGCCATGCGCGGGCGGCAAGCGCGCGCTCGTATGGGTTGAGTGGGGGACCCCATGGCATGGGGTGGGTGAGCGCGGAGGCGTACATGCCGCAAATGTCGTAAACGTAGAGGGGCTCTCTGAGTATTCCAAGATATGTAGGGTAGCATCTTCCACCGCGGATGCTGGCGCGCACGTAATCGTATAGTTCGTGCGAGGGAGCGAGGAGGTCGGGACCGAGGTTGCTACGGGCGGGCTGCTCTGCTCGGAAGACTATCTGCCTGAAGATGGCATGTGAGTTGGATGATATGGTTGGACGCTGGAAGACGTTGAAGCTGGCGTCTGTGAGACCTACCGCGTCACGCACGAAGGAGGCGTAGGAGTCGCGCAGCTTGTTGACCAGCTCGGCGGTGACCTGCACGTCTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTGGTAGGCGCAGCATCCCTTTTCTACGGGTAGCGCGTATGCCTGCGCGGCCTTCCGGCATGACCAGCATGAAGGGCACGAGCTGCTTCCCAAAGGCCCCCATCCAAGTATAGGTCTCTACATCGTAGGTGACAAAGAGACGCTCGGTGCGAGGATGCGAGCCGATCGGGAAGAACTGGATCTCCCGCCACCAATTGGAGGAGTGGCTATTGATGTGGTGAAAGTAGAAGTCCCTGCGACGGGCCGAACACTCGTGCTGGCTTTTGTAAAAACGTGCGCAGTACTGGCAGCGGTGCACGGGCTGTACATCCTGCACGAGGTTGACCTGACGACCGCGCACAAGGAAGCAGAGTGGGAATTTGAGCCCCTCGCCTGGCGGGTTTGGCTGGTGGTCTTCTACTTCGGCTGCTTGTCCTTGACCGTCTGGCTGCTCGAGGGGAGTTACGGTGGATCGGACCACCACGCCGCGCGAGCCCAAAGTCCAGATGTCCGCGCGCGGCGGTCGGAGCTTGATGACAACATCGCGCAGATGGGAGCTGTCCATGGTCTGGAGCTCCCGCGGCGTCAGGTCAGGCGGGAGCTCCTGCAGGTTTACCTCGCATAGACGGGTCAGGGCGCGGGCTAGATCCAGGTGATACCTAATTTCCAGGGGCTGGTTGGTGGCGGCGTCGATGGCTTGCAAGAGGCCGCATCCCCGCGGCGCGACTACGGTACCGCGCGGCGGGCGGTGGGCCGCGGGGGTGTCCTTGGATGATGCATCTAAAAGCGGTGACGCGGGCGAGCCCCCGGAGGTAGGGGGGGCTCCGGACCCGCCGGGAGAGGGGGCAGGGGCACGTCGGCGCCGCGCGCGGGCAGGAGCTGGTGCTGCGCGCGTAGGTTGCTGGCGAACGCGACGACGCGGCGGTTGATCTCCTGAATCTGGCGCCTCTGCGTGAAGACGACGGGCCCGGTGAGCTTGAACCTGAAAGAGAGTTCGACAGAATCAATTTCGGTGTCGTTGACGGCGGCCTGGCGCAAAATCTCCTGCACGTCTCCTGAGTTGTCTTGATAGGCGATCTCGGCCATGAACTGCTCGATCTCTTCCTCCTGGAGATCTCCGCGTCCGGCTCGCTCCACGGTGGCGGCGAGGTCGTTGGAAATGCGGGCCATGAGCTGCGAGAAGGCGTTGAGGCCTCCCTCGTTCCAGACGCGGCTGTAGACCACGCCCCCTTCGGCATCGCGGGCGCGCATGACCACCTGCGCGAGATTGAGCTCCACGTGCCGGGCGAAGACGGCGTAGTTTCGCAGGCGCTGAAAGAGGTAGTTGAGGGTGGTGGCGGTGTGTTCTGCCACGAAGAAGTACATAACCCAGCGTCGCAACGTGGATTCGTTGATAATTGTTGTGTAGGTACTCCGCCGCCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGCGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGACGGCGGATGGTCGACAGAAGCACCATGTCCTTGGGTCCGGCCTGCTGAATGCGCAGGCGGTCGGCCATGCCCCAGGCTTCGTTTTGACATCGGCGCAGGTCTTTGTAGTAGTCTTGCATGAGCCTTTCTACCGGCACTTCTTCTTCTCCTTCCTCTTGTCCTGCATCTCTTGCATCTATCGCTGCGGCGGCGGCGGAGTTTGGCCGTAGGTGGCGCCCTCTTCCTCCCATGCGTGTGACCCCGAAGCCCCTCATCGGCTGAAGCAGGGCTAGGTCGGCGACAACGCGCTCGGCTAATATGGCCTGCTGCACCTGCGTGAGGGTAGACTGGAAGTCATCCATGTCCACAAAGCGGTGGTATGCGCCCGTGTTGATGGTGTAAGTGCAGTTGGCCATAACGGACCAGTTAACGGTCTGGTGACCCGGCTGCGAGAGCTCGGTGTACCTGAGACGCGAGTAAGCCCTCGAGTCAAATACGTAGTCGTTGCAAGTCCGCACCAGGTACTGGTATCCCACCAAAAAGTGCGGCGGCGGCTGGCGGTAGAGGGGCCAGCGTAGGGTGGCCGGGGCTCCGGGGGCGAGATCTTCCAACATAAGGCGATGATATCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCTGGCCGGTCAGGCGCGCGCAATCGTTGACGCTCTAGCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGGTTCGAGCCCCGTATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGTGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGCTGCTGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCAGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTCGCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCGGGACCCCCGGTTCGAGTCTCGGACCGGCCGGACTGCGGCGAACGGGGGTTTGCCTCCCCGTCATGCAAGACCCCGCTTGCAAATTCCTCCGGAAACAGGGACGAGCCCCTTTTTTGCTTTTCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCTCCTCAGCAGCGGCAAGAGCAAGAGCAGCGGCAGACATGCAGGGCACCCTCCCCTCCTCCTACCGCGTCAGGAGGGGCGACATCCGCGGTTGACGCGGCAGCAGATGGTGATTACGAACCCCCGCGGCGCCGGGCCCGGCACTACCTGGACTTGGAGGAGGGCGAGGGCCTGGCGCGGCTAGGAGCGCCCTCTCCTGAGCGGCACCCAAGGGTGCAGCTGAAGCGTGATACGCGTGAGGCGTACGTGCCGCGGCAGAACCTGTTTCGCGACCGCGAGGGAGAGGAGCCCGAGGAGATGCGGGATCGAAAGTTCCACGCAGGGCGCGAGCTGCGGCATGGCCTGAATCGCGAGCGGTTGCTGCGCGAGGAGGACTTTGAGCCCGACGCGCGAACCGGGATTAGTCCCGCGCGCGCACACGTGGCGGCCGCCGACCTGGTAACCGCATACGAGCAGACGGTGAACCAGGAGATTAACTTTCAAAAAAGCTTTAACAACCACGTGCGTACGCTTGTGGCGCGCGAGGAGGTGGCTATAGGACTGATGCATCTGTGGGACTTTGTAAGCGCGCTGGAGCAAAACCCAAATAGCAAGCCGCTCATGGCGCAGCTGTTCCTTATAGTGCAGCACAGCAGGGACAACGAGGCATTCAGGGATGCGCTGCTAAACATAGTAGAGCCCGAGGGCCGCTGGCTGCTCGATTTGATAAACATCCTGCAGAGCATAGTGGTGCAGGAGCGCAGCTTGAGCCTGGCTGACAAGGTGGCCGCCATCAACTATTCCATGCTTAGCCTGGGCAAGTTTTACGCCCGCAAGATATACCATACCCCTTACGTTCCCATAGACAAGGAGGTAAAGATCGAGGGGTTCTACATGCGCATGGCGCTGAAGGTGCTTACCTTGAGCGACGACCTGGGCGTTTATCGCAACGAGCGCATCCACAAGGCCGTGAGCGTGAGCCGGCGGCGCGAGCTCAGCGACCGCGAGCTGATGCACAGCCTGCAAAGGGCCCTGGCTGGCACGGGCAGCGGCGATAGAGAGGCCGAGTCCTACTTTGACGCGGGCGCTGACCTGCGCTGGGCCCCAAGCCGACGCGCCCTGGAGGCAGCTGGGGCCGGACCTGGGCTGGCGGTGGCACCCGCGCGCGCTGGCAACGTCGGCGGCGTGGAGGAATATGACGAGGACGATGAGTACGAGCCAGAGGACGGCGAGTACTAAGCGGTGATGTTTCTGATCAGATGATGCAAGACGCAACGGACCCGGCGGTGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCCTTAACTCCACGGACGACTGGCGCCAGGTCATGGACCGCATCATGTCGCTGACTGCGCGCAATCCTGACGCGTTCCGGCAGCAGCCGCAGGCCAACCGGCTCTCCGCAATTCTGGAAGCGGTGGTCCCGGCGCGCGCAAACCCCACGCACGAGAAGGTGCTGGCGATCGTAAACGCGCTGGCCGAAAACAGGGCCATCCGGCCCGACGAGGCCGGCCTGGTCTACGACGCGCTGCTTCAGCGCGTGGCTCGTTACAACAGCGGCAACGTGCAGACCAACCTGGACCGGCTGGTGGGGGATGTGCGCGAGGCCGTGGCGCAGCGTGAGCGCGCGCAGCAGCAGGGCAACCTGGGCTCCATGGTTGCACTAAACGCCTTCCTGAGTACACAGCCCGCCAACGTGCCGCGGGGACAGGAGGACTACACCAACTTTGTGAGCGCACTGCGGCTAATGGTGACTGAGACACCGCAAAGTGAGGTGTACCAGTCTGGGCCAGACTATTTTTTCCAGACCAGTAGACAAGGCCTGCAGACCGTAAACCTGAGCCAGGCTTTCAAAAACTTGCAGGGGCTGTGGGGGGTGCGGGCTCCCACAGGCGACCGCGCGACCGTGTCTAGCTTGCTGACGCCCAACTCGCGCCTGTTGCTGCTGCTAATAGCGCCCTTCACGGACAGTGGCAGCGTGTCCCGGGACACATACCTAGGTCACTTGCTGACACTGTACCGCGAGGCCATAGGTCAGGCGCATGTGGACGAGCATACTTTCCAGGAGATTACAAGTGTCAGCCGCGCGCTGGGGCAGGAGGACACGGGCAGCCTGGAGGCAACCCTAAACTACCTGCTGACCAACCGGCGGCAGAAGATCCCCTCGTTGCACAGTTTAAACAGCGAGGAGGAGCGCATTTTGCGCTACGTGCAGCAGAGCGTGAGCCTTAACCTGATGCGCGACGGGGTAACGCCCAGCGTGGCGCTGGACATGACCGCGCGCAACATGGAACCGGGCATGTATGCCTCAAACCGGCCGTTTATCAACCGCCTAATGGACTACTTGCATCGCGCGGCCGCCGTGAACCCCGAGTATTTCACCAATGCCATCTTGAACCCGCACTGGCTACCGCCCCCTGGTTTCTACACCGGGGGATTCGAGGTGCCCGAGGGTAACGATGGATTCCTCTGGGACGACATAGACGACAGCGTGTTTTCCCCGCAACCGCAGACCCTGCTAGAGTTGCAACAGCGCGAGCAGGCAGAGGCGGCGCTGCGAAAGGAAAGCTTCCGCAGGCCAAGCAGCTTGTCCGATCTAGGCGCTGCGGCCCCGCGGTCAGATGCTAGTAGCCCATTTCCAAGCTTGATAGGGTCTCTTACCAGCACTCGCACCACCCGCCCGCGCCTGCTGGGCGAGGAGGAGTACCTAAACAACTCGCTGCTGCAGCCGCAGCGCGAAAAAAACCTGCCTCCGGCATTTCCCAACAACGGGATAGAGAGCCTAGTGGACAAGATGAGTAGATGGAAGACGTACGCGCAGGAGCACAGGGACGTGCCAGGCCCGCGCCCGCCCACCCGTCGTCAAAGGCACGACCGTCAGCGGGGTCTGGTGTGGGAGGACGATGACTCGGCAGACGACAGCAGCGTCCTGGATTTGGGAGGGAGTGGCAACCCGTTTGCGCACCTTCGCCCCAGGCTGGGGAGAATGTTTTAAAAAAAAAAAAGCATGATGCAAAATAAAAAACTCACCAAGGCCATGGCACCGAGCGTTGGTTTTCTTGTATTCCCCTTAGTATGCGGCGCGCGGCGATGTATGAGGAAGGTCCTCCTCCCTCCTACGAGAGTGTGGTGAGCGCGGCGCCAGTGGCGGCGGCGCTGGGTTCTCCCTTCGATGCTCCCCTGGACCCGCCGTTTGTGCCTCCGCGGTACCTGCGGCCTACCGGGGGGAGAAACAGCATCCGTTACTCTGAGTTGGCACCCCTATTCGACACCACCCGTGTGTACCTGGTGGACAACAAGTCAACGGATGTGGCATCCCTGAACTACCAGAACGACCACAGCAACTTTCTGACCACGGTCATTCAAAACAATGACTACAGCCCGGGGGAGGCAAGCACACAGACCATCAATCTTGACGACCGGTCGCACTGGGGCGGCGACCTGAAAACCATCCTGCATACCAACATGCCAAATGTGAACGAGTTCATGTTTACCAATAAGTTTAAGGCGCGGGTGATGGTGTCGCGCTTGCCTACTAAGGACAATCAGGTGGAGCTGAAATACGAGTGGGTGGAGTTCACGCTGCCCGAGGGCAACTACTCCGAGACCATGACCATAGACCTTATGAACAACGCGATCGTGGAGCACTACTTGAAAGTGGGCAGACAGAACGGGGTTCTGGAAAGCGACATCGGGGTAAAGTTTGACACCCGCAACTTCAGACTGGGGTTTGACCCCGTCACTGGTCTTGTCATGCCTGGGGTATATACAAACGAAGCCTTCCATCCAGACATCATTTTGCTGCCAGGATGCGGGGTGGACTTCACCCACAGCCGCCTGAGCAACTTGTTGGGCATCCGCAAGCGGCAACCCTTCCAGGAGGGCTTTAGGATCACCTACGATGATCTGGAGGGTGGTAACATTCCCGCACTGTTGGATGTGGACGCCTACCAGGCGAGCTTGAAAGATGACACCGAACAGGGCGGGGGTGGCGCAGGCGGCAGCAACAGCAGTGGCAGCGGCGCGGAAGAGAACTCCAACGCGGCAGCCGCGGCAATGCAGCCGGTGGAGGACATGAACGATCATGCCATTCGCGGCGACACCTTTGCCACACGGGCTGAGGAGAAGCGCGCTGAGGCCGAAGCAGCGGCCGAAGCTGCCGCCCCCGCTGCGCAACCCGAGGTCGAGAAGCCTCAGAAGAAACCGGTGATCAAACCCCTGACAGAGGACAGCAAGAAACGCAGTTACAACCTAATAAGCAATGACAGCACCTTCACCCAGTACCGCAGCTGGTACCTTGCATACAACTACGGCGACCCTCAGACCGGAATCCGCTCATGGACCCTGCTTTGCACTCCTGACGTAACCTGCGGCTCGGAGCAGGTCTACTGGTCGTTGCCAGACATGATGCAAGACCCCGTGACCTTCCGCTCCACGCGCCAGATCAGCAACTTTCCGGTGGTGGGCGCCGAGCTGTTGCCCGTGCACTCCAAGAGCTTCTACAACGACCAGGCCGTCTACTCCCAACTCATCCGCCAGTTTACCTCTCTGACCCACGTGTTCAATCGCTTTCCCGAGAACCAGATTTTGGCGCGCCCGCCAGCCCCCACCATCACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACGCTACCGCTGCGCAACAGCATCGGAGGAGTCCAGCGAGTGACCATTACTGACGCCAGACGCCGCACCTGCCCCTACGTTTACAAGGCCCTGGGCATAGTCTCGCCGCGCGTCCTATCGAGCCGCACTTTTTGAGCAAGCATGTCCATCCTTATATCGCCCAGCAATAACACAGGCTGGGGCCTGCGCTTCCCAAGCAAGATGTTTGGCGGGGCCAAGAAGCGCTCCGACCAACACCCAGTGCGCGTGCGCGGGCACTACCGCGCGCCCTGGGGCGCGCACAAACGCGGCCGCACTGGGCGCACCACCGTCGATGACGCCATCGACGCGGTGGTGGAGGAGGCGCGCAACTACACGCCCACGCCGCCACCAGTGTCCACAGTGGACGCGGCCATTCAGACCGTGGTGCGCGGAGCCCGGCGCTATGCTAAAATGAAGAGACGGCGGAGGCGCGTAGCACGTCGCCACCGCCGCCGACCCGGCACTGCCGCCCAACGCGCGGCGGCGGCCCTGCTTAACCGCGCACGTCGCACCGGCCGACGGGCGGCCATGCGGGCCGCTCGAAGGCTGGCCGCGGGTATTGTCACTGTGCCCCCCAGGTCCAGGCGACGAGCGGCCGCCGCAGCAGCCGCGGCCATTAGTGCTATGACTCAGGGTCGCAGGGGCAACGTGTATTGGGTGCGCGACTCGGTTAGCGGCCTGCGCGTGCCCGTGCGCACCCGCCCCCCGCGCAACTAGATTGCAAGAAAAAACTACTTAGACTCGTACTGTTGTATGTATCCAGCGGCGGCGGCGCGCAACGAAGCTATGTCCAAGCGCAAAATCAAAGAAGAGATGCTCCAGGTCATCGCGCCGGAGATCTATGGCCCCCCGAAGAAGGAAGAGCAGGATTACAAGCCCCGAAAGCTAAAGCGGGTCAAAAAGAAAAAGAAAGATGATGATGATGAACTTGACGACGAGGTGGAACTGCTGCACGCTACCGCGCCCAGGCGACGGGTACAGTGGAAAGGTCGACGCGTAAAACGTGTTTTGCGACCCGGCACCACCGTAGTCTTTACGCCCGGTGAGCGCTCCACCCGCACCTACAAGCGCGTGTATGATGAGGTGTACGGCGACGAGGACCTGCTTGAGCAGGCCAACGAGCGCCTCGGGGAGTTTGCCTACGGAAAGCGGCATAAGGACATGCTGGCGTTGCCGCTGGACGAGGGCAACCCAACACCTAGCCTAAAGCCCGTAACACTGCAGCAGGTGCTGCCCGCGCTTGCACCGTCCGAAGAAAAGCGCGGCCTAAAGCGCGAGTCTGGTGACTTGGCACCCACCGTGCAGCTGATGGTACCCAAGCGCCAGCGACTGGAAGATGTCTTGGAAAAAATGACCGTGGAACCTGGGCTGGAGCCCGAGGTCCGCGTGCGGCCAATCAAGCAGGTGGCGCCGGGACTGGGCGTGCAGACCGTGGACGTTCAGATACCCACTACCAGTAGCACCAGTATTGCCACCGCCACAGAGGGCATGGAGACACAAACGTCCCCGGTTGCCTCAGCGGTGGCGGATGCCGCGGTGCAGGCGGTCGCTGCGGCCGCGTCCAAGACCTCTACGGAGGTGCAAACGGACCCGTGGATGTTTCGCGTTTCAGCCCCCCGGCGCCCGCGCCGTTCGAGGAAGTACGGCGCCGCCAGCGCGCTACTGCCCGAATATGCCCTACATCCTTCCATTGCGCCTACCCCCGGCTATCGTGGCTACACCTACCGCCCCAGAAGACGAGCAACTACCCGACGCCGAACCACCACTGGAACCCGCCGCCGCCGTCGCCGTCGCCAGCCCGTGCTGGCCCCGATTTCCGTGCGCAGGGTGGCTCGCGAAGGAGGCAGGACCCTGGTGCTGCCAACAGCGCGCTACCACCCCAGCATCGTTTAAAAGCCGGTCTTTGTGGTTCTTGCAGATATGGCCCTCACCTGCCGCCTCCGTTTCCCGGTGCCGGGATTCCGAGGAAGAATGCACCGTAGGAGGGGCATGGCCGGCCACGGCCTGACGGGCGGCATGCGTCGTGCGCACCACCGGCGGCGGCGCGCGTCGCACCGTCGCATGCGCGGCGGTATCCTGCCCCTCCTTATTCCACTGATCGCCGCGGCGATTGGCGCCGTGCCCGGAATTGCATCCGTGGCCTTGCAGGCGCAGAGACACTGATTAAAAACAAGTTGCATGTGGAAAAATCAAAATAAAAAGTCTGGACTCTCACGCTCGCTTGGTCCTGTAACTATTTTGTAGAATGGAAGACATCAACTTTGCGTCTCTGGCCCCGCGACACGGCTCGCGCCCGTTCATGGGAAACTGGCAAGATATCGGCACCAGCAATATGAGCGGTGGCGCCTTCAGCTGGGGCTCGCTGTGGAGCGGCATTAAAAATTTCGGTTCCACCGTTAAGAACTATGGCAGCAAGGCCTGGAACAGCAGCACAGGCCAGATGCTGAGGGATAAGTTGAAAGAGCAAAATTTCCAACAAAAGGTGGTAGATGGCCTGGCCTCTGGCATTAGCGGGGTGGTGGACCTGGCCAACCAGGCAGTGCAAAATAAGATTAACAGTAAGCTTGATCCCCGCCCTCCCGTAGAGGAGCCTCCACCGGCCGTGGAGACAGTGTCTCCAGAGGGGCGTGGCGAAAAGCGTCCGCGCCCCGACAGGGAAGAAACTCTGGTGACGCAAATAGACGAGCCTCCCTCGTACGAGGAGGCACTAAAGCAAGGCCTGCCCACCACCCGTCCCATCGCGCCCATGGCTACCGGAGTGCTGGGCCAGCACACACCCGTAACGCTGGACCTGCCTCCCCCCGCCGACACCCAGCAGAAACCTGTGCTGCCAGGCCCGACCGCCGTTGTTGTAACCCGTCCTAGCCGCGCGTCCCTGCGCCGCGCCGCCAGCGGTCCGCGATCGTTGCGGCCCGTAGCCAGTGGCAACTGGCAAAGCACACTGAACAGCATCGTGGGTCTGGGGGTGCAATCCCTGAAGCGCCGACGATGCTTCTGATAGCTAACGTGTCGTATGTGTGTCATGTATGCGTCCATGTCGCCGCCAGAGGAGCTGCTGAGCCGCCGCGCGCCCGCTTTCCAAGATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACAGACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCTGTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAACCTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACAGAAATTAATCATGCAGCTGGGAGAGTCCTAAAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCAACTACTGAGGCAGCCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGCTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTATAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCAGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAAAGAAGCAAGCAACATCAACAACAGCTGCCGCCATGGGCTCCAGTGAGCAGGAACTGAAAGCCATTGTCAAAGATCTTGGTTGTGGGCCATATTTTTTGGGCACCTATGACAAGCGCTTTCCAGGCTTTGTTTCTCCACACAAGCTCGCCTGCGCCATAGTCAATACGGCCGGTCGCGAGACTGGGGGCGTACACTGGATGGCCTTTGCCTGGAACCCGCACTCAAAAACATGCTACCTCTTTGAGCCCTTTGGCTTTTCTGACCAGCGACTCAAGCAGGTTTACCAGTTTGAGTACGAGTCACTCCTGCGCCGTAGCGCCATTGCTTCTTCCCCCGACCGCTGTATAACGCTGGAAAAGTCCACCCAAAGCGTACAGGGGCCCAACTCGGCCGCCTGTGGACTATTCTGCTGCATGTTTCTCCACGCCTTTGCCAACTGGCCCCAAACTCCCATGGATCACAACCCCACCATGAACCTTATTACCGGGGTACCCAACTCCATGCTCAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGAGACACTTTCAATAAAGGCAAATGCTTTTATTTGTACACTCTCGGGTGATTATTTACCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGCGCGTGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAAAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATAAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCGTGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTTCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGATGCTTGTAGGTCACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTCAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGTTTGAAGTTCGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAGCGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCACACTCAGCGGGTTCATCACCGTAATTTCACTTTCCGCTTCGCTGGGCTCTTCCTCTTCCTCTTGCGTCCGCATACCACGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACTGTGCGCTTACCTCCTTTGCCATGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGATTACCTCTGGTGATGGCGGGCGCTCGGGCTTGGGAGAAGGGCGCTTCTTTTTCTTCTTGGGCGCAATGGCCAAATCCGCCGCCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCGTCTTGTGATGAGTCTTCCTCGTCCTCGGACTCGATACGCCGCCTCATCCGCTTTTTTGGGGGCGCCCGGGGAGGCGGCGGCGACGGGGACGGGGACGACACGTCCTCCATGGTTGGGGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGAAGGACAGCCTAACCGCCCCCTCTGAGTTCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGACCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACAACGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACGAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCCTTGCCTACGAACGCCACCTATTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAACGGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCAACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACGAGAAGCGCGCGGCAAACGCTCTGCAACAGGAAAACAGCGAAAATGAAAGTCACTCTGGAGTGTTGGTGGAACTCGAGGGTGACAACGCGCGCCTAGCCGTACTAAAACGCAGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTCATGAGCACAGTCATGAGTGAGCTGATCGTGCGCCGTGCGCAGCCCCTGGAGAGGGATGCAAATTTGCAAGAACAAACAGAGGAGGGCCTACCCGCAGTTGGCGACGAGCAGCTAGCGCGCTGGCTTCAAACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAACTAATGATGGCCGCAGTGCTCGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCGCAAGCTAGAGGAAACATTGCACTACACCTTTCGACAGGGCTACGTACGCCAGGCCTGCAAGATCTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAACCGCCTTGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTATGCTACACCTGGCAGACGGCCATGGGCGTTTGGCAGCAGTGCTTGGAGGAGTGCAACCTCAAGGAGCTGCAGAAACTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGACATCATTTTCCCCGAACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAGAACTTTAGGAACTTTATCCTAGAGCGCTCAGGAATCTTGCCCGCCACCTGCTGTGCACTTCCTAGCGACTTTGTGCCCATTAAGTACCGCGAATGCCCTCCGCCGCTTTGGGGCCACTGCTACCTTCTGCAGCTAGCCAACTACCTTGCCTACCACTCTGACATAATGGAAGACGTGAGCGGTGACGGTCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCTGGTTTGCAATTCGCAGCTGCTTAACGAAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCTAATGCGGAGCTTACCGCCTGCGTCATTACCCAGGGCCACATTCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGTTTACTTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGGACATGATGGAAGACTGGGAGAGCCTAGACGAGGAAGCTTCCGAGGTCGAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATCGGCAACCGGTTCCAGCATGGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCCGTTCGCCGACCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCCAAGCAGCCGCCGCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCATGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCGTCATCTCTACAGCCCATACTGCACCGGCGGCAGCGGCAGCAACAGCAGCGGCCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGCGAGCTTAGAAACAGGATTTTTCCCACTCTGTATGCTATATTTCAACAGAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGATCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGTAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCCAGCACCTGTTGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATACGCGCCCACCGAAACCGAATTCTCCTGGAACAGGCGGCTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCAGGGTATAACTCACCTGACAATCAGAGGGCGAGGTATTCAGCTCAACGACGAGTCGGTGAGCTCCTCGCTTGGTCTCCGTCCGGACGGGACATTTCAGATCGGCGGCGCCGGCCGCTCTTCATTCACGCCTCGTCAGGCAATCCTAACTCTGCAGACCTCGTCCTCTGAGCCGCGCTCTGGAGGCATTGGAACTCTGCAATTTATTGAGGAGTTTGTGCCATCGGTCTACTTTAACCCCTTCTCGGGACCTCCCGGCCACTATCCGGATCAATTTATTCCTAACTTTGACGCGGTAAAGGACTCGGCGGACGGCTACGACTGAATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACCTGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCCGGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGAGGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAGCTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCTGCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTGATTTGCAACTGTCCTAACCCTGGATTACATCAAGATCCTCTAGTTAATGTCAGGTCGCCTAAGTCGATTAACTAGAGTACCCGGGGATCTTATTCCCTTTAACTAATAAAAAAAAATAATAAAGCATCACTTACTTAAAATCAGTTAGCAAATTTCTGTCCAGTTTATTCAGCAGCACCTCCTTGCCCTCCTCCCAGCTCTGGTATTGCAGCTTCCTCCTGGCTGCAAACTTTCTCCACAATCTAAATGGAATGTCAGTTTCCTCCTGTTCCTGTCCATCCGCACCCACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCATATGACACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAATGGGTTTCAAGAGAGTCCCCCTGGGGTACTCTCTTTGCGCCTATCCGAACCTCTAGTTACCTCCAATGGCATGCTTGCGCTCAAAATGGGCAACGGCCTCTCTCTGGACGAGGCCGGCAACCTTACCTCCCAAAATGTAACCACTGTGAGCCCACCTCTCAAAAAAACCAAGTCAAACATAAACCTGGAAATATCTGCACCCCTCACAGTTACCTCAGAAGCCCTAACTGTGGCTGCCGCCGCACCTCTAATGGTCGCGGGCAACACACTCACCATGCAATCACAGGCCCCGCTAACCGTGCACGACTCCAAACTTAGCATTGCCACCCAAGGACCCCTCACAGTGTCAGAAGGAAAGCTAGCCCTGCAAACATCAGGCCCCCTCACCACCACCGATAGCAGTACCCTTACTATCACTGCCTCACCCCCTCTAACTACTGCCACTGGTAGCTTGGGCATTGACTTGAAAGAGCCCATTTATACACAAAATGGAAAACTAGGACTAAAGTACGGGGCTCCTTTGCATGTAACAGACGACCTAAACACTTTGACCGTAGCAACTGGTCCAGGTGTGACTATTAATAATACTTCCTTGCAAACTAAAGTTACTGGAGCCTTGGGTTTTGATTCACAAGGCAATATGCAACTTAATGTAGCAGGAGGACTAAGGATTGATTCTCAAAACAGACGCCTTATACTTGATGTTAGTTATCCGTTTGATGCTCAAAACCAACTAAATCTAAGACTAGGACAGGGCCCTCTTTTTATAAACTCAGCCCACAACTTGGATATTAACTACAACAAAGGCCTTTACTTGTTTACAGCTTCAAACAATTCCAAAAAGCTTGAGGTTAACCTAAGCACTGCCAAGGGGTTGATGTTTGACGCTACAGCCATAGCCATTAATGCAGGAGATGGGCTTGAATTTGGTTCACCTAATGCACCAAACACAAATCCCCTCAAAACAAAAATTGGCCATGGCCTAGAATTTGATTCAAACAAGGCTATGGTTCCTAAACTAGGAACTGGCCTTAGTTTTGACAGCACAGGTGCCATTACAGTAGGAAACAAAAATAATGATAAGCTAACTTTGTGGACCACACCAGCTCCATCTCCTAACTGTAGACTAAATGCAGAGAAAGATGCTAAACTCACTTTGGTCTTAACAAAATGTGGCAGTCAAATACTTGCTACAGTTTCAGTTTTGGCTGTTAAAGGCAGTTTGGCTCCAATATCTGGAACAGTTCAAAGTGCTCATCTTATTATAAGATTTGACGAAAATGGAGTGCTACTAAACAATTCCTTCCTGGACCCAGAATATTGGAACTTTAGAAATGGAGATCTTACTGAAGGCACAGCCTATACAAACGCTGTTGGATTTATGCCTAACCTATCAGCTTATCCAAAATCTCACGGTAAAACTGCCAAAAGTAACATTGTCAGTCAAGTTTACTTAAACGGAGACAAAACTAAACCTGTAACACTAACCATTACACTAAACGGTACACAGGAAACAGGAGACACAACTCCAAGTGCATACTCTATGTCATTTTCATGGGACTGGTCTGGCCACAACTACATTAATGAAATATTTGCCACATCCTCTTACACTTTTTCATACATTGCCCAAGAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTCAATTGCAGAAAATTTCAAGTCATTTTTCATTCAGTAGTATAGCCCCACCACCACATAGCTTATACAGATCACCGTACCTTAATCAAACTCACAGAACCCTAGTATTCAACCTGCCACCTCCCTCCCAACACACAGAGTACACAGTCCTTTCTCCCCGGCTGGCCTTAAAAAGCATCATATCATGGGTAACAGACATATTCTTAGGTGTTATATTCCACACGGTTTCCTGTCGAGCCAAACGCTCATCAGTGATATTAATAAACTCCCCGGGCAGCTCACTTAAGTTCATGTCGCTGTCCAGCTGCTGAGCCACAGGCTGCTGTCCAACTTGCGGTTGCTTAACGGGCGGCGAAGGAGAAGTCCACGCCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATAAGGCGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTATACACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTTAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGCGCGGGTTTCTGTCTCAAAAGGAGGTAGACGATCCCTACTGTACGGAGTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTCATATTTCCTGAAGCAAAACCAGGTGCGGGCGTGACAAACAGATCTGCGTCTCCGGTCTCGCCGCTTAGATCGCTCTGTGTAGTAGTTGTAGTATATCCACTCTCTCAAAGCATCCAGGCGCCCCCTGGCTTCGGGTTCTATGTAAACTCCTTCATGCGCCGCTGCCCTGATAACATCCACCACCGCAGAATAAGCCACACCCAGCCAACCTACACATTCGTTCTGCGAGTCACACACGGGAGGAGCGGGAAGAGCTGGAAGAACCATGTTTTTTTTTTTATTCCAAAAGATTATCCAAAACCTCAAAATGAAGATCTATTAAGTGAACGCGCTCCCCTCCGGTGGCGTGGTCAAACTCTACAGCCAAAGAACAGATAATGGCATTTGTAAGATGTTGCACAATGGCTTCCAAAAGGCAAACGGCCCTCACGTCCAAGTGGACGTAAAGGCTAAACCCTTCAGGGTGAATCTCCTCTATAAACATTCCAGCACCTTCAACCATGCCCAAATAATTCTCATCTCGCCACCTTCTCAATATATCTCTAAGCAAATCCCGAATATTAAGTCCGGCCATTGTAAAAATCTGCTCCAGAGCGCCCTCCACCTTCAGCCTCAAGCAGCGAATCATGATTGCAAAAATTCAGGTTCCTCACAGACCTGTATAAGATTCAAAAGCGGAACATTAACAAAAATACCGCGATCCCGTAGGTCCCTTCGCAGGGCCAGCTGAACATAATCGTGCAGGTCTGCACGGACCAGCGCGGCCACTTCCCCGCCAGGAACCATGACAAAAGAACCCACACTGATTATGACACGCATACTCGGAGCTATGCTAACCAGCGTAGCCCCGATGTAAGCTTGTTGCATGGGCGGCGATATAAAATGCAAGGTGCTGCTCAAAAAATCAGGCAAAGCCTCGCGCAAAAAAGAAAGCACATCGTAGTCATGCTCATGCAGATAAAGGCAGGTAAGCTCCGGAACCACCACAGAAAAAGACACCATTTTTCTCTCAAACATGTCTGCGGGTTTCTGCATAAACACAAAATAAAATAACAAAAAAACATTTAAACATTAGAAGCCTGTCTTACAACAGGAAAAACAACCCTTATAAGCATAAGACGGACTACGGCCATGCCGGCGTGACCGTAAAAAAACTGGTCACCGTGATTAAAAAGCACCACCGACAGCTCCTCGGTCATGTCCGGAGTCATAATGTAAGACTCGGTAAACACATCAGGTTGATTCACATCGGTCAGTGCTAAAAAGCGACCGAAATAGCCCGGGGGAATACATACCCGCAGGCGTAGAGACAACATTACAGCCCCCATAGGAGGTATAACAAAATTAATAGGAGAGAAAAACACATAAACACCTGAAAAACCCTCCTGCCTAGGCAAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGG TATATTATTGATGAT,.

In some embodiments, an adenovirus-derived vector, optionally relatingto a replication defective adenovirus, comprises a sequence with atleast 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% identity toSEQ. ID. NO: 3 or a sequence generated from SEQ. ID. NO: 3 byalternative codon replacements. In various embodiments, theadenovirus-derived vectors described herein have a deletion in the E2bregion, and optionally, in the E1 region, the deletion conferring avariety of advantages to the use of the vectors in immunotherapy asdescribed herein.

Certain regions within the adenovirus genome serve essential functionsand may need to be substantially conserved when constructing thereplication defective adenovirus vectors of the invention. These regionsare further described in Lauer (Lauer et al. Natural variation amonghuman adenoviruses: genome sequence and annotation of human adenovirusserotype 1. Journal of General Virology (2004), 85, 2615-2625), Leza(Leza et al. Cellular Transcription Factor Binds to Adenovirus EarlyRegion Promotersandtoa CyclicAMP ResponseElement. Journal OF VIROLOGY,August 1988, p. 3003-3013), and Miralles (Miralles et al. The AdenovirusInverted Terminal Repeat Functions as an Enhancer in a Cell-free System.THE JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 264, No. 18, Issue of June 25,pp. 10763-10772,1983), which are each herein incorporate by reference intheir entirety. Recombinant nucleic acid vectors comprising a sequencewith identity values of at least 75%, 80%, 85%, 90%, 95%, 98%, 99%,99.5%, 99.8%, 99.9% to a portion of SEQ. ID. NO: 3 are within the boundsof the invention.

First generation, E1-deleted Adenovirus subtype 5 (Ad5)-based vectors,although promising platforms for use as cancer vaccines, are impeded inactivity by naturally occurring or induced Ad-specific neutralizingantibodies. Ad5-based vectors with deletions of the E1 and the E2bregions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase andthe pre-terminal protein, by virtue of diminished late phase viralprotein expression, provide an opportunity to avoid immunologicalclearance and induce more potent immune responses against the encodedtumor antigen transgene in Ad-immune hosts. Indeed, multiple homologousimmunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding a variant of thetumor antigen CEA, induced CEA-specific cell-mediated immune (CMI)responses with antitumor activity in mice despite the presence ofpre-existing or induced Ad5-neutralizing antibody. In a phase I/IIstudy, cohorts of patients with advanced colorectal cancer wereimmunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specificCMI responses were observed despite the presence of pre-existing Ad5immunity in a majority (61.3%) of patients. Importantly, there wasminimal toxicity, and overall patient survival (48% at 12 months) wassimilar regardless of pre-existing Ad5 neutralizing antibody titers. Theresults demonstrated that, in cancer patients, the novel Ad5 [E1-, E2b-]gene delivery platform generates significant CMI responses to the tumorantigen CEA in the setting of both naturally acquired andimmunization-induced Ad5 specific immunity. CEA antigen specific CMI canbe, for example, greater than 10, 20, 30, 40, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 5000, 10000, or more IFN-γ spot formingcells (SFC) per 10⁶ peripheral blood mononuclear cells (PBMC). Thus, themethods and compositions of the invention relate to a recombinantnucleic acid vector, wherein the recombinant nucleic acid vectorcomprises a replication defective adenovirus vector, and wherein uponadministration to a human, the composition is capable of inducing animmune response directed towards cells expressing CEA antigen in saidhuman. The immune response may be induced even in the presence ofpreexisting immunity against Ad5. In some embodiments, the immuneresponse is raised in a human subject with a preexisting inverse Ad5neutralizing antibody titer of greater than 50, 100, 150, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 6000, 7000, 8000, 9000, 1000, 12000, 15000 or higher. The immuneresponse may comprise a cell-mediated immunity and/or a humoral immunityas described herein. The immune response may be measured by one or moreof intracellular cytokine staining (ICS), ELISpot, proliferation assays,cytotoxic T cell assays including chromium release or equivalent assays,and gene expression analysis using any number of polymerase chainreaction (PCR) or RT-PCR based assays, as described herein and to theextent they are available to a person skilled in the art, as well as anyother suitable assays known in the art for measuring immune response.

While cancer immunotherapy achieved by delivering tumor-associatedantigens (TAA) provides survival benefits, limitations to thesestrategies exist and more immunologically potent vaccines are needed. Toaddress the low immunogenicity of self-tumor antigens, a variety ofadvanced, multi-component vaccination strategies includingco-administration of adjuvants and immune stimulating cytokines areprovided. The invention relates to recombinant viral vectors thatinherently provide innate pro-inflammatory signals, while simultaneouslyengineered to express the antigen of interest. Of particular interestare adenovirus serotype-5 (Ad5)-based immunotherapeutics that have beenrepeatedly used in humans to induce robust T cell-mediated immune (CMI)responses, all while maintaining an extensive safety profile. Inaddition, Ad5 vectors can be reliably manufactured in large quantitiesand are stable for storage and delivery for outpatient administration.Nonetheless, a major obstacle to the use of first generation(E1-deleted) Ad5-based vectors is the high frequency of pre-existinganti-adenovirus type 5 neutralizing antibodies. These antibodies can bepresent in a potential vaccinee due to either prior wild type adenovirusinfection and/or induction of adenovirus neutralizing antibodies byrepeated injections with Ad5-based vaccines, each resulting ininadequate immune stimulation against the target TAA.

Attempts to overcome anti-Ad immunity have included use of alternativeAd serotypes and/or alternations in the Ad5 viral capsid protein eachwith limited success and the potential for significantly alteringbiodistribution of the resultant vaccines. Therefore, a completely novelapproach was attempted by further reducing the expression of viralproteins from the E1 deleted Ad5 vectors, proteins known to be targetsof pre-existing Ad immunity. Specifically, a novel recombinant Ad5platform has been described with deletions in the early 1 (E1) generegion and additional deletions in the early 2b (E2b) gene region (Ad5[E1-, E2b-]). Deletion of the E2b region (that encodes DNA polymeraseand the pre-terminal protein) results in decreased viral DNA replicationand late phase viral protein expression. This vector platform has beenpreviously reported to successfully induce CMI responses in animalmodels of cancer and infectious disease and more importantly, thisrecombinant Ad5 gene delivery platform overcomes the barrier of Ad5immunity and can be used in the setting of pre-existing and/orvector-induced Ad immunity thus enabling multiple homologousadministrations of the vaccine. We have constructed and tested an Ad5[E1-, E2b-] platform containing a gene insert for the tumor antigencarcinoembryonic antigen (CEA) with a modification that enhances T cellresponses (Ad5 [E1-, E2b-]-CEA(6D) and is used in various embodiments ofthe invention for therapies raising an immune response against CEA.Multiple immunizations with this Ad5 platform induced CEA-specific CMIresponses with antitumor activity despite the presence of existing Ad5immunity in mice. The results of a first-in-man, phase I/II clinicaltrial demonstrate safety and immunogenicity in humans. A dose escalationof the Ad5 [E1-, E2b-]-CEA(6D) vector in advanced stage colorectalcancer patients demonstrates that CMI can be induced without asubstantial effect on clinical outcome relative to the existence ofpre-existing Ad5-immunity.

CEA as Target for Immune Response

CEA represents an attractive target antigen for immunotherapy since itis over-expressed in nearly all colorectal cancers and pancreaticcancers, and is also expressed by some lung and breast cancers, anduncommon tumors such as medullary thyroid cancer, but is not expressedin other cells of the body except for low-level expression ingastrointestinal epithelium (Berinstein, N. L. 2002. Carcinoembryonicantigen as a target for therapeutic anticancer vaccines: a review. JClin Oncol 20:2197-2207.). CEA contains epitopes that may be recognizedin an MHC restricted fashion by T cells.

Members of the CEA gene family are subdivided into three subgroups basedon sequence similarity, developmental expression patterns and theirbiological functions: the CEA-related Cell Adhesion Molecule (CEACAM)subgroup containing twelve genes (CEACAM1, CEACAM3-CEACAM8, CEACAM16 andCEACAM18-CEACAM21), the Pregnancy Specific Glycoprotein (PSG) subgroupcontaining eleven closely related genes (PSG1-PSG11) and a subgroup ofeleven pseudogenes (CEACAMP1-CEACAMP11) (Zhou et al. 2000; Gray-Owen andBlumberg 2006; FIGS. 1.4-1.5). Most members of the CEACAM subgroup havesimilar structures consist of an extracellular Ig-like domains composedof a single N-terminal V-set domain, with structural homology to theimmunoglobulin variable domains, followed by varying numbers of C2-setdomains of A or B subtypes, a transmembrane domain and a cytoplasmicdomain (Beauchemin et al. 1999; Kammerer et al., 2007). There are twomembers of CEACAM subgroup (CEACAM16 and CEACAM20) that show a fewexceptions in the organization of their structures. CEACAM16 containstwo Ig-like V-type domains at its N and C termini and CEACAM20 containsa truncated Ig-like V-type 1 domain (FIG. 1.4). The CEACAM molecules canbe anchored to the cell surface via their transmembrane domains (CEACAM5thought CEACAM8) or directly linked to glycophosphatidylinositol (GPI)lipid moiety (CEACAM5, CEACAM18 thought CEACAM21).

Members of CEA family are known to be expressed in different cell typesand have a wide range of biological functions. CEACAMs are foundprominently on most epithelial cells and are present on differentleucocytes. In humans, CEACAM1, the ancestor member of CEA family, isexpressed on the apical side of epithelial and endothelial cells as wellas on lymphoid and myeloid cells. CEACAM1 mediates cell-cell adhesionthrough homophilic (CEACAM1 to CEACAM1) as well as heterophilic (e.g.CEACAM1 to CEACAM5) interactions (Kuespert et al. 2006; Gray-Owen andBlumberg 2006). In addition, CEACAM1 is involved in many otherbiological processes, such as angiogenesis, cell migration, and immunefunctions (Gray-Owen and Blumberg 2006). CEACAM3 and CEACAM4 expressionis largely restricted to granulocytes, and they are able to conveyuptake and destruction of several bacterial pathogens includingNeisseria, Moraxella, and Haemophilus species (Schmitter et al. 2004).

Thus, in various embodiments, compositions and methods of the inventionrelate to raising an immune response against a CEA, selected from thegroup consisting of CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6,CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, CEACAM21,PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG7, PSG8, PSG9, and PSG11. Animmune response may be raised against cells, e.g. cancer cells,expressing or overexpressing one or more of the CEAs, using the methodsand compositions of the invention. In some embodiments, theoverexpression of the one or more CEAs in such cancer cells is over 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold or more compared tonon-cancer cells.

Further, compositions and methods of the invention, in variousembodiments, take advantage of human cytolytic T cells (CTLs),specifically those that recognize CEA epitopes which bind to selectedMHC molecules, e.g. HLA-A2, A3, and A24. Individuals expressing MHCmolecules of certain serotypes, e.g. HLA-A2, A3, and A24 may be selectedfor therapy using the methods and compositions of the invention. Forexample, individuals expressing MHC molecules of certain serotypes, e.g.HLA-A2, A3, and A24, may be selected for a therapy including raising animmune response against CEA, using the methods and compositionsdescribed herein.

In various embodiments, these T cells can be generated by in vitrocultures using antigen-presenting cells pulsed with the epitope ofinterest to stimulate peripheral blood mononuclear cells. In addition, Tcell lines can also be generated after stimulation with CEA latex beads,CEA protein-pulsed plastic adherent peripheral blood mononuclear cells,or DCs sensitized with CEA RNA. T cells can also be generated frompatients immunized with a vaccine vector encoding CEA immunogen. HLAA2-presented peptides from CEA can further be found in primarygastrointestinal tumors. In various embodiments, the invention relatesto an HLA A2 restricted epitope of CEA, CAP-1, a nine amino acidsequence (YLSGANLNL; SEQ. ID. NO.: 4), with ability to stimulate CTLsfrom cancer patients immunized with vaccine-CEA. Cap-1(6D) (YLSGADLNL;SEQ. ID. NO.: 5) is a peptide analog of CAP-1. Its sequence includes aheteroclitic (nonanchor position) mutation, resulting in an amino acidchange from Asn to Asp, enhancing recognition by the T-cell receptor.The Asn to Asp mutation appears to not cause any change in the bindingof the peptide to HLA A2. Compared with the non-mutated CAP-1 epitope,Cap-1(6D) can enhance the sensitization of CTLs by 100 to 1,000 times(Tsang, K. Y., Zhu, M., Nieroda, C. A., Correale, P., Zaremba, S.,Hamilton, J. M., Cole, D., Lam, C., and Schlom, J. 1997. Phenotypicstability of a cytotoxic T-cell line directed against an immunodominantepitope of human carcinoembryonic antigen. Clin Cancer Res 3:2439-2449;Zaremba, S., Barzaga, E., Zhu, M., Soares, N., Tsang, K. Y., and Schlom,J. 1997. Identification of an enhancer agonist cytotoxic T lymphocytepeptide from human carcinoembryonic antigen. Cancer Res 57:4570-4577;Tangri, S., Ishioka, G. Y., Huang, X., Sidney, J., Southwood, S., Fikes,J., and Sette, A. 2001. Structural features of peptide analogs of humanhistocompatibility leukocyte antigen class I epitopes that are morepotent and immunogenic than wild-type peptide. J Exp Med 194:833-846).CTL lines can be elicited from peripheral blood mononuclear cells ofhealthy volunteers by in vitro sensitization to the Cap-1(6D) peptide,but not significantly to the CAP-1 peptide. These cell lines can lysehuman tumor cells expressing endogenous CEA. Thus, polypeptide sequencescomprising CAP-1 or CAP-1(6D), nucleic acid sequences encoding suchsequences, an adenovirus vectors; for example replication defectiveadenovirus vectors, comprising such nucleic acid sequences are withinthe bounds of the invention.

Clinical Trials of Vaccines Targeting CEA

Although CEA can serve as a T cell target and CEA-specific T cellprecursors exist in humans, their frequency is quite low (<1/100,000).The invention, in various embodiments relates to increasing theirnumbers sufficiently and harnessing them to lyse CEA-expressing tumorcells. Several phase I clinical trials assessing various vaccineapproaches for activating CEA-specific T cells were performed. Earlystudies were based on vaccination using protein with adjuvant (Samanci,A., Yi, Q., Fagerberg, J., Strigård, K., Smith, G., Rudén, U., Wahren,B., and Mellstedt, H. 1998. Pharmacological administration ofgranulocyte/macrophage-colony-stimulating factor is of significantimportance for the induction of a strong humoral and cellular responsein patients immunized with recombinant carcinoembryonic antigen. CancerImmunol Immunother 47:131-142), or in the form of anti-idiotype vaccines(Foon, K. A., John, W. J., Chakraborty, M., Das, R., Teitelbaum, A.,Garrison, J., Kashala, O., Chatterjee, S. K., andBhattacharya-Chatterjee, M. 1999. Clinical and immune responses inresected colon cancer patients treated with anti-idiotype monoclonalantibody vaccine that mimics the carcinoembryonic antigen. J Clin Oncol17:2889-2885; Foon, K. A., John, W. J., Chakraborty, M., Sherratt, A.,Garrison, J., Flett, M., and Bhattacharya-Chatterjee, M. 1997. Clinicaland immune responses in advanced colorectal cancer patients treated withanti-idiotype monoclonal antibody vaccine that mimics thecarcinoembryonic antigen. Clin Cancer Res 3:1267-1276). CEA-specific Tcell proliferative responses were observed in a substantial proportionof the patients in these studies; although the magnitude of the immuneresponses was modest. In order to improve upon these results, poxvirusesengineered to express tumor antigens have been developed (Paoletti, E.1996. Applications of pox virus vectors to vaccination: an update. ProcNatl Acad Sci USA 93:11349-11353; Cox, W. I., Tartaglia, J., andPaoletti, E. 1993. Induction of cytotoxic T lymphocytes by recombinantcanarypox (ALVAC) and attenuated vaccinia (NYVAC) viruses expressing theHIV-1 envelope glycoprotein. Virology 195:845-850; Taylor, J.,Trimarchi, C., Weinberg, R., Languet, B., Guillemin, F., Desmettre, P.,and Paoletti, E. 1991. Efficacy studies on a canarypox-rabiesrecombinant virus. Vaccine 9:190-193). Both the vaccinia poxvirus andALVAC, a variant of the canary poxvirus, have been geneticallyengineered to contain the genes that encode for various antigens, sothat when the virus is injected into the body, it enters the host'scells and induces them to produce the antigen of interest. Because thesevectors do not integrate into the genome, there is no risk ofinsertional mutagenesis, and expression of viral products and transgenesis transient, lasting only 10 to 14 days. Various immunization schemesagainst CEA utilizing such vectors were previously studied, but showedlimited to no clinical response.

The compositions and methods of the invention, in various embodiments,provide adenovirus based vectors expressing a variant CEA forimmunization against CEA. These vectors can raise an immune responseagainst CEA. Further, in various embodiments, the composition andmethods of the invention lead to clinical responses, such as altereddisease progression or life expectancy.

Ad5 Vaccines

Adenoviruses are a family of DNA viruses characterized by anicosohedral, non-enveloped capsid containing a linear double-strandedgenome. Of the human Ads, none are associated with any neoplasticdisease, and only cause relatively mild, self-limiting illness inimmunocompetent individuals. The first genes expressed by the virus arethe E1 genes, which act to initiate high-level gene expression from theother Ad5 gene promoters present in the wild type genome. Viral DNAreplication and assembly of progeny virions occur within the nucleus ofinfected cells, and the entire life cycle takes about 36 hr with anoutput of approximately 10⁴ virions per cell. The wild type Ad5 genomeis approximately 36 kb, and encodes genes that are divided into earlyand late viral functions, depending on whether they are expressed beforeor after DNA replication. The early/late delineation is nearly absolute,since it has been demonstrated that super-infection of cells previouslyinfected with an Ad5 results in lack of late gene expression from thesuper-infecting virus until after it has replicated its own genome.Without bound by theory, this is likely due to a replication dependentcis-activation of the Ad5 major late promoter (MLP), preventing lategene expression (primarily the Ad5 capsid proteins) until replicatedgenomes are present to be encapsulated. The composition and methods ofthe invention take advantage of feature in the development of advancedgeneration Ad vectors/vaccines.

Ad5 Vectors

First generation, or E1-deleted adenovirus vectors Ad5 [E1-] areconstructed such that a transgene replaces only the E1 region of genes.Typically, about 90% of the wild-type Ad5 genome is retained in thevector. Ad5 [E1-] vectors have a decreased ability to replicate andcannot produce infectious virus after infection of cells not expressingthe Ad5 E1 genes. The recombinant Ad5 [E1-] vectors are propagated inhuman cells (typically 293 cells) allowing for Ad5 [E1-] vectorreplication and packaging. Ad5 [E1-] vectors have a number of positiveattributes; one of the most important is their relative ease for scaleup and cGMP production. Currently, well over 220 human clinical trialsutilize Ad5 [E1-] vectors, with more than two thousand subjects giventhe virus sc, im, or iv. Additionally, Ad5 vectors do not integrate;their genomes remain episomal. Generally, for vectors that do notintegrate into the host genome, the risk for insertional mutagenesisand/or germ-line transmission is extremely low if at all. ConventionalAd5 [E1-] vectors have a carrying capacity that approaches 7 kb.

Ad5 [E1-] Vectors Used as a Cancer Vaccine:

Arthur et. al. demonstrated that Ad5 [E1-] vectors encoding a variety ofantigens could efficiently transduce 95% of ex vivo exposed DC's to hightiters of the vector (Arthur, J. F., Butterfield, L. H., Roth, M. D.,Bui, L. A., Kiertscher, S. M., Lau, R., Dubinett, S., Glaspy, J.,McBride, W. H., and Economou, J. S. 1997. A comparison of gene transfermethods in human dendritic cells. Cancer Gene Ther 4:17-25).Importantly, increasing levels of foreign gene expression were noted inthe DC with increasing multiplicities of infection (MOI) with thevector, a finding repeated by others, as well as reproduced in ourpreliminary studies (Diao, J., Smythe, J. A., Smyth, C., Rowe, P. B.,and Alexander, I. E. 1999. Human PBMC-derived dendritic cells transducedwith an adenovirus vector induce cytotoxic T-lymphocyte responsesagainst a vector-encoded antigen in vitro. Gene Ther 6:845-853). It hasbeen demonstrated that DC infected with Ad5 [E1-] vectors encoding avariety of antigens (including the tumor antigens MART-1, MAGE-A4,DF3/MUC1, p53, hugp100 melanoma antigen, polyoma virus middle-T antigen)have the propensity to induce antigen specific CTL responses, have anenhanced antigen presentation capacity, and have an improved ability toinitiate T-cell proliferation in mixed lymphocyte reactions (Arthur, J.F., Butterfield, L. H., Roth, M. D., Bui, L. A., Kiertscher, S. M., Lau,R., Dubinett, S., Glaspy, J., McBride, W. H., and Economou, J. S. 1997.A comparison of gene transfer methods in human dendritic cells. CancerGene Ther 4:17-25; Diao, J., Smythe, J. A., Smyth, C., Rowe, P. B., andAlexander, I. E. 1999. Human PBMC-derived dendritic cells transducedwith an adenovirus vector induce cytotoxic T-lymphocyte responsesagainst a vector-encoded antigen in vitro. Gene Ther 6:845-853;Brossart, P., Goldrath, A. W., Butz, E. A., Martin, S., and Bevan, M. J.1997. Virus-mediated delivery of antigenic epitopes into dendritic cellsas a means to induce CTL. J Immunol 158:3270-3276; Butterfield, L. H.,Jilani, S. M., Chakraborty, N. G., Bui, L. A., Ribas, A., Dissette, V.B., Lau, R., Gamradt, S. C., Glaspy, J. A., McBride, W. H., et al. 1998.Generation of melanoma-specific cytotoxic T lymphocytes by dendriticcells transduced with a MART-1 adenovirus. J Immunol 161:5607-5613;Bregni, M., Shammah, S., Malaffo, F., Di Nicola, M., Milanesi, M.,Magni, M., Matteucci, P., Ravagnani, F., Jordan, C. T., Siena, S., etal. 1998. Adenovirus vectors for gene transduction into mobilized bloodCD34+ cells. Gene Ther 5:465-472; Dietz, A. B., and Vuk-Pavlovic, S.1998. High efficiency adenovirus-mediated gene transfer to humandendritic cells. Blood 91:392-398; Ishida, T., Chada, S., Stipanov, M.,Nadaf, S., Ciernik, F. I., Gabrilovich, D. I., and Carbone, D. P. 1999.Dendritic cells transduced with wild-type p53 gene elicit potentanti-tumour immune responses. Clin Exp Immunol 117:244-251; Ribas, A.,Butterfield, L. H., McBride, W. H., Jilani, S. M., Bui, L. A., Vollmer,C. M., Lau, R., Dissette, V. B., Hu, B., Chen, A. Y., et al. 1997.Genetic immunization for the melanoma antigen MART-1/Melan-A usingrecombinant adenovirus-transduced murine dendritic cells. Cancer Res57:2865-2869). Immunization of animals with DC's previously transducedby Ad5 vectors encoding tumor specific antigens has been demonstrated toresult in significant levels of protection for the animals whenchallenged with tumor cells expressing the respective antigen (Wan, Y.,Bramson, J., Carter, R., Graham, F., and Gauldie, J. 1997. Dendriticcells transduced with an adenoviral vector encoding a modeltumor-associated antigen for tumor vaccination. Hum Gene Ther8:1355-1363; Wan, Y., Emtage, P., Foley, R., Carter, R., and Gauldie, J.1999. Murine dendritic cells transduced with an adenoviral vectorexpressing a defined tumor antigen can overcome anti-adenovirusneutralizing immunity and induce effective tumor regression. Int J Oncol14:771-776). Interestingly, intra-tumoral injection of Ads encoding IL-7was less effective than injection of DCs transduced with IL-7 encodingAd5 vectors at inducing antitumor immunity, further heightening theinterest in ex vivo transduction of DCs by Ad5 vectors (Miller, P. W.,Sharma, S., Stolina, M., Butterfield, L. H., Luo, J., Lin, Y.,Dohadwala, M., Batra, R. K., Wu, L., Economou, J. S., et al. 2000.Intratumoral administration of adenoviral interleukin 7 gene-modifieddendritic cells augments specific antitumor immunity and achieves tumoreradication. Hum Gene Ther 11:53-65). Ex vivo DC transduction strategieshave also been used to attempt to induce tolerance in recipient hosts,for example, by Ad5 mediated delivery of the CTLA4Ig into DCs, blockinginteractions of the DCs CD80 with the CD28 molecule present on T-cells(Lu, L., Gambotto, A., Lee, W. C., Qian, S., Bonham, C. A., Robbins, P.D., and Thomson, A. W. 1999. Adenoviral delivery of CTLA4Ig into myeloiddendritic cells promotes their in vitro tolerogenicity and survival inallogeneic recipients. Gene Ther 6:554-563).

Ad5 vector capsid interactions with DCs in and of themselves may triggerseveral beneficial responses, which may be enhancing the propensity ofDCs to present antigens encoded by Ad5 vectors. For example, immatureDCs, though specialized in antigen uptake, are relatively inefficienteffectors of T-cell activation. DC maturation coincides with theenhanced ability of DCs to drive T-cell immunity. In some instances, thecompositions and methods of the invention take advantage of an Ad5infection resulting in direct induction of DC maturation (Rea, D.,Schagen, F. H., Hoeben, R. C., Mehtali, M., Havenga, M. J., Toes, R. E.,Melief, C. J., and Offringa, R. 1999. Adenoviruses activate humandendritic cells without polarization toward a T-helper type 1-inducingsubset. J Virol 73:10245-10253; Hirschowitz, E. A., Weaver, J. D.,Hidalgo, G. E., and Doherty, D. E. 2000. Murine dendritic cells infectedwith adenovirus vectors show signs of activation. Gene Ther7:1112-1120). Studies of immature bone marrow derived DCs from micesuggest that Ad vector infection of immature bone marrow derived DCsfrom mice resulted may upregulate cell surface markers normallyassociated with DC maturation (MHC I and II, CD40, CD80, CD86, andICAM-1) as well as down-regulation of CD11c, an integrin known to bedown regulated upon myeloid DC maturation. In some instances, Ad vectorinfection triggers IL-12 production by DCs, a marker of DC maturation(Hirschowitz, E. A., Weaver, J. D., Hidalgo, G. E., and Doherty, D. E.2000. Murine dendritic cells infected with adenovirus vectors show signsof activation. Gene Ther 7:1112-1120). Without being bound by theory,these events may possibly be due to Ad5 triggered activation of NF-κBpathways (Hirschowitz, E. A., Weaver, J. D., Hidalgo, G. E., andDoherty, D. E. 2000. Murine dendritic cells infected with adenovirusvectors show signs of activation. Gene Ther 7:1112-1120; Loser, P.,Jennings, G. S., Strauss, M., and Sandig, V. 1998. Reactivation of thepreviously silenced cytomegalovirus major immediate-early promoter inthe mouse liver: involvement of NF-κB. J Virol 72:180-190; Morelli, A.E., Larregina, A. T., Ganster, R. W., Zahorchak, A. F., Plowey, J. M.,Takayama, T., Logar, A. J., Robbins, P. D., Falo, L. D., and Thomson, A.W. 2000. Recombinant adenovirus induces maturation of dendritic cellsvia an NF-κB-dependent pathway. J Virol 74:9617-9628). Mature DCs can beefficiently transduced by Ad vectors, and did not lose their functionalpotential to stimulate the proliferation of naive T-cells at lower MOI,as demonstrated by mature CD83+ human DC (derived from peripheral bloodmonocytes. However, mature DCs may also be less infectable than immatureones (Rea, D., Schagen, F. H., Hoeben, R. C., Mehtali, M., Havenga, M.J., Toes, R. E., Melief, C. J., and Offringa, R. 1999. Adenovirusesactivate human dendritic cells without polarization toward a T-helpertype 1-inducing subset. J Virol 73:10245-10253; Jonuleit, H., Tüting,T., Steitz, J., Bruck, J., Giesecke, A., Steinbrink, K., Knop, J., andEnk, A. H. 2000. Efficient transduction of mature CD83+ dendritic cellsusing recombinant adenovirus suppressed T cell stimulatory capacity.Gene Ther 7:249-254). Modification of capsid proteins can be used as astrategy to optimize infection of DC by Ad vectors, as well as enhancingfunctional maturation, for example using the CD40L receptor as a viralvector receptor, rather than using the normal CAR receptor infectionmechanisms (Tillman, B. W., Hayes, T. L., DeGruijl, T. D., Douglas, J.T., and Curiel, D. T. 2000. Adenoviral vectors targeted to CD40 enhancethe efficacy of dendritic cell-based vaccination against humanpapillomavirus 16-induced tumor cells in a murine model. Cancer Res60:5456-5463).

Most dramatically, when the potential of non-viral vectors to induceanti-HIV immune responses was directly compared to Ad5 based vectoringsystems, the Ad5 based systems were found to be far superior (Shiver, J.W., Fu, T. M., Chen, L., Casimiro, D. R., Davies, M. E., Evans, R. K.,Zhang, Z. Q., Simon, A. J., Trigona, W. L., Dubey, S. A., et al. 2002.Replication-incompetent adenoviral vaccine vector elicits effectiveanti-immunodeficiency-virus immunity. Nature 415:331-335). For example,in Ad5 naïve primate models, vaccination with a Ad5 [E1-] expressing theHIV gag was superior in protecting the animals from SHIV infections ascompared to similar efforts utilizing naked DNA vaccines expressingHIV-gag. Thus, viral vectors can be superior to naked DNA approaches(Shiver, J. W., Fu, T. M., Chen, L., Casimiro, D. R., Davies, M. E.,Evans, R. K., Zhang, Z. Q., Simon, A. J., Trigona, W. L., Dubey, S. A.,et al. 2002. Replication-incompetent adenoviral vaccine vector elicitseffective anti-immunodeficiency-virus immunity. Nature 415:331-335).Combined strategies (building upon their clinical experiences with nakedDNA-gag vectors alone) using naked DNA-gag vaccines as a primingvaccination, followed by boosting with the Ad5 [E1-]-gag vaccine furtherimproved T cell responses in human trials than those previously notedwith the DNA-HIV-gag encoding vector alone (HVTN working group meeting,Alexandria, Va.: 2002).

In summary, Ad5 vectors appear to offer a unique opportunity to allowfor high level and efficient transduction of TAAs such as CEA. One ofthe major problems facing Ad5 based vectors is the high propensity ofpre-existing immunity to Ads in the human population, and how this maypreclude the use of conventional, Ad5 [E1-] deleted (first generationAds) in most human populations, for any additional vaccine application.

Ad5 [E1-, E2B-]-CEA(6D) Vaccine: The Use of Ad5 [E1-, E2b-] Vaccines toOvercome the Challenge of Pre-Existing Anti-Ad5 Immunity

Studies in humans and animals have demonstrated that pre-existingimmunity against Ad5 can be an inhibitory factor to commercial use ofAd-based vaccines (Yang, Z. Y., Wyatt, L. S., Kong, W. P., Moodie, Z.,Moss, B., and Nabel, G. J. 2003. Overcoming immunity to a viral vaccineby DNA priming before vector boosting. J Virol 77:799-803; Casimiro, D.R., Chen, L., Fu, T. M., Evans, R. K., Caulfield, M. J., Davies, M. E.,Tang, A., Chen, M., Huang, L., Harris, V., et al. 2003. Comparativeimmunogenicity in rhesus monkeys of DNA plasmid, recombinant vacciniavirus, and replication-defective adenovirus vectors expressing a humanimmunodeficiency virus type 1 gag gene. J Virol 77:6305-6313). Thepreponderance of humans have antibody against Ad5, the most widely usedsubtype for human vaccines, with two-thirds of humans studied havinglympho-proliferative responses against Ad5 (Chirmule, N., Propert, K.,Magosin, S., Qian, Y., Qian, R., and Wilson, J. 1999. Immune responsesto adenovirus and adeno-associated virus in humans. Gene Ther6:1574-1583). This pre-existing immunity can inhibit immunization orre-immunization using typical Ad5 vaccines and may preclude theimmunization of a vaccinee against a second antigen, using an Ad5vector, at a later time. Overcoming the problem of pre-existinganti-vector immunity has been a subject of intense investigation.Investigations using alternative human (non-Ad5 based) Ad5 subtypes oreven non-human forms of Ad5 have been examined. Even if these approachessucceed in an initial immunization, subsequent vaccinations may beproblematic due to immune responses to the novel Ad5 subtype. To avoidthe Ad5 immunization barrier, and improve upon the limited efficacy offirst generation Ad5 [E1-] vectors to induce optimal immune responses,various embodiments of the invention relate to a next generation Ad5vector based vaccine platform. The new Ad5 platform has additionaldeletions in the E2b region, removing the DNA polymerase and thepreterminal protein genes. The Ad5 [E1-, E2b-] platform has an expandedcloning capacity that is sufficient to allow inclusion of many possiblegenes (Hartigan-O'Connor, D., Barjot, C., Salvatori, G., andChamberlain, J. S. 2002. Generation and growth of gutted adenoviralvectors. Methods Enzymol 346:224-246). Ad5 [E1-, E2b-] vectors have upto about 12 kb gene-carrying capacity as compared to the 7 kb capacityof Ad5 [E1-] vectors, providing space for multiple genes if needed. Insome embodiments, an insert of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 kb is introduced into an Ad5 vector, such as the Ad5 [E1-, E2b-]vector. Deletion of the E2b region confers advantageous immuneproperties on the Ad5 vectors of the invention, often eliciting potentimmune responses to target transgene antigens while minimizing theimmune responses to Ad viral proteins.

In various embodiments, Ad5 [E1-, E2b-] vectors of the invention inducea potent CMI, as well as antibodies against the vector expressed vaccineantigens even in the presence of Ad immunity (Harui, A., Roth, M. D.,Kiertscher, S. M., Mitani, K., and Basak, S. K. 2004. Vaccination withhelper-dependent adenovirus enhances the generation oftransgene-specific CTL. Gene Ther 11:1617-1626). Ad5 [E1-, E2b-] vectorsalso have reduced adverse reactions as compared to Ad5 [E1-] vectors, inparticular the appearance of hepatotoxicity and tissue damage (Hodges,B. L., Serra, D., Hu, H., Begy, C. A., Chamberlain, J. S., andAmalfitano, A. 2000. Multiply deleted [E1, polymerase-, and pTP-]adenovirus vector persists despite deletion of the preterminal protein.J Gene Med 2:250-259; Morral, N., Parks, R. J., Zhou, H., Langston, C.,Schiedner, G., Quinones, J., Graham, F. L., Kochanek, S., and Beaudet,A. L. 1998. High doses of a helper-dependent adenoviral vector yieldsupraphysiological levels of alpha1-antitrypsin with negligibletoxicity. Hum Gene Ther 9:2709-2716; DelloRusso, C., Scott, J. M.,Hartigan-O'Connor, D., Salvatori, G., Barjot, C., Robinson, A. S.,Crawford, R. W., Brooks, S. V., and Chamberlain, J. S. 2002. Functionalcorrection of adult mdx mouse muscle using gutted adenoviral vectorsexpressing full-length dystrophin. Proc Natl Acad Sci USA99:12979-12984; Reddy, P. S., Sakhuja, K., Ganesh, S., Yang, L., Kayda,D., Brann, T., Pattison, S., Golightly, D., Idamakanti, N., Pinkstaff,A., et al. 2002. Sustained human factor VIII expression in hemophilia Amice following systemic delivery of a gutless adenoviral vector. MolTher 5:63-73). A key aspect of these Ad5 vectors is that expression ofAd late genes is greatly reduced (Hodges, B. L., Serra, D., Hu, H.,Begy, C. A., Chamberlain, J. S., and Amalfitano, A. 2000. Multiplydeleted [E1, polymerase-, and pTP-] adenovirus vector persists despitedeletion of the preterminal protein. J Gene Med 2:250-259; Amalfitano,A., Hauser, M. A., Hu, H., Serra, D., Begy, C. R., and Chamberlain, J.S. 1998. Production and characterization of improved adenovirus vectorswith the E1, E2b, and E3 genes deleted. J Virol 72:926-933;Hartigan-O'Connor, D., Kirk, C. J., Crawford, R., Mulé, J. J., andChamberlain, J. S. 2001. Immune evasion by muscle-specific geneexpression in dystrophic muscle. Mol Ther 4:525-533). For example,production of the capsid fiber proteins could be detected in vivo forAd5 [E1-] vectors, while fiber expression was ablated from Ad5 [E1-,E2b-] vector vaccines (Hu, H., Serra, D., and Amalfitano, A. 1999.Persistence of an [E1-, polymerase-] adenovirus vector despitetransduction of a neoantigen into immune-competent mice. Hum Gene Ther10:355-364; DelloRusso, C., Scott, J. M., Hartigan-O'Connor, D.,Salvatori, G., Barjot, C., Robinson, A. S., Crawford, R. W., Brooks, S.V., and Chamberlain, J. S. 2002. Functional correction of adult mdxmouse muscle using gutted adenoviral vectors expressing full-lengthdystrophin. Proc Natl Acad Sci USA 99:12979-12984; Reddy, P. S.,Sakhuja, K., Ganesh, S., Yang, L., Kayda, D., Brann, T., Pattison, S.,Golightly, D., Idamakanti, N., Pinkstaff, A., et al. 2002. Sustainedhuman factor VIII expression in hemophilia A mice following systemicdelivery of a gutless adenoviral vector. Mol Ther 5:63-73). The innateimmune response to wild type Ad is complex. Proteins deleted from theAd5 [E1-, E2b-] vectors generally play an important role. Specifically,Ad5 [E1-, E2b-] vectors with deletions of preterminal protein or DNApolymerase display reduced inflammation during the first 24 to 72 hoursfollowing injection compared to Ad5 [E1-] vectors. In variousembodiments, the lack of Ad5 gene expression renders infected cellsinvisible to anti-Ad activity and permits infected cells to express thetransgene for extended periods of time, which develops immunity to thetarget.

Various embodiments of the invention contemplate increasing thecapability for the Ad5 [E1-, E2b-] vectors to transduce dendritic cells,improving antigen specific immune responses in the vaccine by takingadvantage of the reduced inflammatory response against Ad5 [E1-, E2b-]vector viral proteins and the resulting evasion of pre-existing Adimmunity.

In some cases, this immune induction may take months. Ad5 [E1-, E2b-]vectors not only are safer than, but appear to be superior to Ad5 [E1-]vectors in regard to induction of antigen specific immune responses,making them much better suitable as a platform to deliver CEA vaccinesthat can result in a clinical response.

Various embodiments of the invention, by taking advantage of the new Ad5[E1-, E2b-] vector system in delivering a long sought-after need for adevelop a therapeutic vaccine against CEA, overcome barriers found withother Ad5 systems and permit the immunization of people who havepreviously been exposed to Ad5.

In various embodiments the compositions and methods of the inventioncomprising an Ad5 [E1-, E2b-] vector CEA vaccine effect of increasedoverall survival (OS) within the bounds of technical safety.

Adenovirus Vectors

Compared to First Generation adenovirus vectors, certain embodiments ofthe Second Generation E2b deleted adenovirus vectors of the presentinvention contain additional deletions in the DNA polymerase gene (pol)and deletions of the pre-terminal protein (pTP). E2b deleted vectorshave up to a 13 kb gene-carrying capacity as compared to the 5 to 6 kbcapacity of First Generation adenovirus vectors, easily providing spacefor nucleic acid sequences encoding any of a variety of target antigens.The E2b deleted adenovirus vectors also have reduced adverse reactionsas compared to First Generation adenovirus vectors (Morral, et al HumGene Ther 9/2709-2716 (1998); Hodges, et al. J Gene Med 2/250-259(2000); DelloRusso, et al. Proc Natl Acad Sci USA 99/12979-12984 (2002);Reddy, et al. Mol Ther 5/63-73 (2002); (Amalfitano and Parks, et al.Curr Gene Ther 2/111-133 (2002); Amalfitano Curr Opin Mol Ther 5/362-366(2003); Everett, et al. Human Gene Ther 14/1715-1726 (2003)) E2b deletedvectors have reduced expression of viral genes (Hodges, et al. J GeneMed 2/250-259 (2000); Amalfitano, et al. J Virol 72/926-933 (1998);Hartigan-O'Connor, et al. Mol Ther 4/525-533 (2001)), and thischaracteristic has been reported to lead to extended transgeneexpression in vivo (Hu, et al. Hum Gene Ther 10/355-364 (1999);DelloRusso, et al. Proc Natl Acad Sci USA 99/12979-12984 (2002); Reddy,et al. Mol Ther 5/63-73 (2002); (Amalfitano and Parks, et al. Curr GeneTher 2/111-133 (2002); Amalfitano Curr Opin Mol Ther 5/362-366 (2003);Everett, et al. Human Gene Ther 14/1715-1726 (2003)).

The innate immune response to wild type Ad can be complex, and itappears that Ad proteins expressed from adenovirus vectors play animportant role (Moorhead, et al. J Virol 73/1046-1053 (1999); Nazir, etal. J Investig Med 53/292-304 (2005); Schaack, et al. Proc Natl Acad SciUSA 101/3124-3129 (2004); Schaack, et al. Viral Immunol 18/79-88 (2005);Kiang, et al. Mol Ther 14/588-598 (2006); Hartman, et al. J Virol81/1796-1812 (2007); Hartman, et al. Virology 358/357-372 (2007)).Specifically, the deletions of pre-terminal protein and DNA polymerasein the E2b deleted vectors appear to reduce inflammation during thefirst 24 to 72 hours following injection, whereas First Generationadenovirus vectors stimulate inflammation during this period (Schaack,et al. Proc Natl Acad Sci USA 101/3124-3129 (2004); Schaack, et al.Viral Immunol 18/79-88 (2005); Kiang, et al. Mol Ther 14/588-598 (2006);Hartman, et al. J Virol 81/1796-1812 (2007); Hartman, et al. Virology358/357-372 (2007)). In addition, it has been reported that theadditional replication block created by E2b deletion also leads to a10,000 fold reduction in expression of Ad late genes, well beyond thatafforded by E1, E3 deletions alone (Amalfitano et al. J. Virol.72/926-933 (1998); Hodges et al. J. Gene Med. 2/250-259 (2000)). Thedecreased levels of Ad proteins produced by E2b deleted adenovirusvectors effectively reduce the potential for competitive, undesired,immune responses to Ad antigens, responses that prevent repeated use ofthe platform in Ad immunized or exposed individuals. The reducedinduction of inflammatory response by Second Generation E2b deletedvectors results in increased potential for the vectors to expressdesired vaccine antigens during the infection of antigen presentingcells (i.e. dendritic cells), decreasing the potential for antigeniccompetition, resulting in greater immunization of the vaccine to thedesired antigen relative to identical attempts with First Generationadenovirus vectors. E2b deleted adenovirus vectors provide an improvedAd-based vaccine candidate that is safer, more effective, and moreversatile than previously described vaccine candidates using FirstGeneration adenovirus vectors.

Thus, first generation, E1-deleted Adenovirus subtype 5 (Ad5)-basedvectors, although promising platforms for use as cancer vaccines, areimpeded in activity by naturally occurring or induced Ad-specificneutralizing antibodies. Without being bound by theory, Ad5-basedvectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]),the latter encoding the DNA polymerase and the pre-terminal protein, forexample by virtue of diminished late phase viral protein expression, mayavoid immunological clearance and induce more potent immune responsesagainst the encoded tumor antigen transgene in Ad-immune hosts. Indeed,multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encodingthe tumor antigen CEA, induced CEA-specific cell-mediated immune (CMI)responses with antitumor activity in mice despite the presence ofpre-existing or induced Ad5-neutralizing antibody. In the present phaseI/II study, cohorts of patients with advanced colorectal cancer wereimmunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specificCMI responses were observed despite the presence of pre-existing Ad5immunity in a majority (61.3%) of patients. Importantly, there wasminimal toxicity, and overall patient survival (48% at 12 months) wassimilar regardless of pre-existing Ad5 neutralizing antibody titers. Theresults demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-]gene delivery platform generates significant CMI responses to the tumorantigen CEA in the setting of both naturally acquired andimmunization-induced Ad5specific immunity.

The present invention contemplates the use of E2b deleted adenovirusvectors, such as those described in U.S. Pat. Nos. 6,063,622; 6,451,596;6,057,158; 6,083,750; and 8,298,549, which are each incorporated hereinby reference in their entirety. The vectors with deletions in the E2bregions in many cases cripple viral protein expression and/or decreasethe frequency of generating replication competent Ad (RCA). Propagationof these E2b deleted adenovirus vectors can be done utilizing cell linesthat express the deleted E2b gene products. The present invention alsoprovides such packaging cell lines; for example E.C7 (formally calledC-7), derived from the HEK-203 cell line (Amalfitano, et al. Proc NatlAcad Sci USA 93/3352-3356 (1996); Amalfitano, et al. Gene Ther 4/258-263(1997)).

Further, the E2b gene products, DNA polymerase and preterminal protein,can be constitutively expressed in E.C7, or similar cells along with theE1 gene products. Transfer of gene segments from the Ad genome to theproduction cell line has immediate benefits: (1) increased carryingcapacity; and, (2) a decreased potential of RCA generation, typicallyrequiring two or more independent recombination events to generate RCA.The E1, Ad DNA polymerase and/or preterminal protein expressing celllines used in the present invention can enable the propagation ofadenovirus vectors with a carrying capacity approaching 13 kb, withoutthe need for a contaminating helper virus [Mitani et al. (1995) Proc.Natl. Acad. Sci. USA 92:3854; Hodges, et al., 2000 J Gene Med 2:250-259;(Amalfitano and Parks, Curr Gene Ther 2/111-133 (2002)]. In addition,when genes critical to the viral life cycle are deleted (e.g., the E2bgenes), a further crippling of Ad to replicate or express other viralgene proteins occurs. This can decrease immune recognition of virallyinfected cells, and allow for extended durations of foreign transgeneexpression.

E1, DNA polymerase, and preterminal protein deleted vectors aretypically unable to express the respective proteins from the E1 and E2bregions. Further, they may show a lack of expression of most of theviral structural proteins. For example, the major late promoter (MLP) ofAd is responsible for transcription of the late structural proteins L1through L5 [Doerfler, In Adenovirus DNA, The Viral Genome and ItsExpression (Martinus Nijhoff Publishing Boston, 1986)]. Though the MLPis minimally active prior to Ad genome replication, the highly toxic Adlate genes are primarily transcribed and translated from the MLP onlyafter viral genome replication has occurred [Thomas and Mathews (1980)Cell 22:523]. This cis-dependent activation of late gene transcriptionis a feature of DNA viruses in general, such as in the growth of polyomaand SV-40. The DNA polymerase and preterminal proteins are important forAd replication (unlike the E4 or protein IX proteins). Their deletioncan be extremely detrimental to adenovirus vector late gene expression,and the toxic effects of that expression in cells such as APCs.

In certain embodiments, the adenovirus vectors contemplated for use inthe present invention include E2b deleted adenovirus vectors that have adeletion in the E2b region of the Ad genome and, optionally, the E1region. In some cases, such vectors do not have any other regions of theAd genome deleted. In another embodiment, the adenovirus vectorscontemplated for use in the present invention include E2b deletedadenovirus vectors that have a deletion in the E2b region of the Adgenome and, optionally, deletions in the E 1 and E3 regions. In somecases, such vectors have no other regions deleted. In a furtherembodiment, the adenovirus vectors contemplated for use in the presentinvention include adenovirus vectors that have a deletion in the E2bregion of the Ad genome and, optionally, deletions in the E1, E3 and,also optionally, partial or complete removal of the E4 regions. In somecases, such vectors have no other deletions. In another embodiment, theadenovirus vectors contemplated for use in the present invention includeadenovirus vectors that have a deletion in the E2b region of the Adgenome and, optionally deletions in the E1 and/or E4 regions. In somecases, such vectors contain no other deletions. In an additionalembodiment, the adenovirus vectors contemplated for use in the presentinvention include adenovirus vectors that have a deletion in the E2a,E2b and/or E4 regions of the Ad genome. In some cases, such vectors haveno other deletions. In one embodiment, the adenovirus vectors for useherein comprise vectors having the E1 and/or DNA polymerase functions ofthe E2b region deleted. In some cases, such vectors have no otherdeletions. In a further embodiment, the adenovirus vectors for useherein have the E1 and/or the preterminal protein functions of the E2bregion deleted. In some cases, such vectors have no other deletions. Inanother embodiment, the adenovirus vectors for use herein have the E1,DNA polymerase and/or the preterminal protein functions deleted. In somecases, such vectors have no other deletions. In one particularembodiment, the adenovirus vectors contemplated for use herein aredeleted for at least a portion of the E2b region and/or the E1 region.In some cases, such vectors are not “gutted” adenovirus vectors. In thisregard, the vectors may be deleted for both the DNA polymerase and thepreterminal protein functions of the E2b region. In an additionalembodiment, the adenovirus vectors for use in the present inventioninclude adenovirus vectors that have a deletion in the E1, E2b and/or100K regions of the adenovirus genome. In one embodiment, the adenovirusvectors for use herein comprise vectors having the E1, E2b and/orprotease functions deleted. In some cases, such vectors have no otherdeletions. In a further embodiment, the adenovirus vectors for useherein have the E1 and/or the E2b regions deleted, while the fiber geneshave been modified by mutation or other alterations (for example toalter Ad tropism). Removal of genes from the E3 or E4 regions may beadded to any of the mentioned adenovirus vectors. In certainembodiments, the adenovirus vector may be a “gutted” adenovirus vector.

The term “E2b deleted”, as used herein, refers to a specific DNAsequence that is mutated in such a way so as to prevent expressionand/or function of at least one E2b gene product. Thus, in certainembodiments, “E2b deleted” is used in relation to a specific DNAsequence that is deleted (removed) from the Ad genome. E2b deleted or“containing a deletion within the E2b region” refers to a deletion of atleast one base pair within the E2b region of the Ad genome. Thus, incertain embodiments, more than one base pair is deleted and in furtherembodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, or 150 base pairs are deleted. In another embodiment, thedeletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 basepairs within the E2b region of the Ad genome. An E2b deletion may be adeletion that prevents expression and/or function of at least one E2bgene product and therefore, encompasses deletions within exons ofencoding portions of E2b-specific proteins as well as deletions withinpromoter and leader sequences. In certain embodiments, an E2b deletionis a deletion that prevents expression and/or function of one or both ofthe DNA polymerase and the preterminal protein of the E2b region. In afurther embodiment, “E2b deleted” refers to one or more point mutationsin the DNA sequence of this region of an Ad genome such that one or moreencoded proteins is non-functional. Such mutations include residues thatare replaced with a different residue leading to a change in the aminoacid sequence that result in a nonfunctional protein.

The term “E1 deleted”, as used herein, refers to a specific DNA sequencethat is mutated in such a way so as to prevent expression and/orfunction of at least one E1 gene product. Thus, in certain embodiments,“E1 deleted” is used in relation to a specific DNA sequence that isdeleted (removed) from the Ad genome. E1 deleted or “containing adeletion within the E1 region” refers to a deletion of at least one basepair within the E1 region of the Ad genome. Thus, in certainembodiments, more than one base pair is deleted and in furtherembodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, or 150 base pairs are deleted. In another embodiment, thedeletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 basepairs within the E1 region of the Ad genome. An E1 deletion may be adeletion that prevents expression and/or function of at least one E1gene product and therefore, encompasses deletions within exons ofencoding portions of E1-specific proteins as well as deletions withinpromoter and leader sequences. In certain embodiments, an E1 deletion isa deletion that prevents expression and/or function of one or both of atrans-acting transcriptional regulatory factor of the E1 region. In afurther embodiment, “E1 deleted” refers to one or more point mutationsin the DNA sequence of this region of an Ad genome such that one or moreencoded proteins is non-functional. Such mutations include residues thatare replaced with a different residue leading to a change in the aminoacid sequence that result in a nonfunctional protein.

As would be understood by the skilled artisan upon reading the presentdisclosure, other regions of the Ad genome can be deleted. Thus to be“deleted” in a particular region of the Ad genome, as used herein,refers to a specific DNA sequence that is mutated in such a way so as toprevent expression and/or function of at least one gene product encodedby that region. In certain embodiments, to be “deleted” in a particularregion refers to a specific DNA sequence that is deleted (removed) fromthe Ad genome in such a way so as to prevent the expression and/or thefunction encoded by that region (e.g., E2b functions of DNA polymeraseor preterminal protein function). “Deleted” or “containing a deletion”within a particular region refers to a deletion of at least one basepair within that region of the Ad genome. Thus, in certain embodiments,more than one base pair is deleted and in further embodiments, at least20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 basepairs are deleted from a particular region. In another embodiment, thedeletion is more than 150, 160, 170, 180, 190, 200, 250, or 300 basepairs within a particular region of the Ad genome. These deletions aresuch that expression and/or function of the gene product encoded by theregion is prevented. Thus deletions encompass deletions within exonsencoding portions of proteins as well as deletions within promoter andleader sequences. In a further embodiment, “deleted” in a particularregion of the Ad genome refers to one or more point mutations in the DNAsequence of this region of an Ad genome such that one or more encodedproteins is non-functional. Such mutations include residues that arereplaced with a different residue leading to a change in the amino acidsequence that result in a nonfunctional protein. Deletions or mutationsin the Ad genome can be within one or more of E1a, E1b, E2a, E2b, E3,E4, L1, L2, L3, L4, L5, TP, POL, IV, and VA regions.

The deleted adenovirus vectors of the present invention can be generatedusing recombinant techniques known in the art (see e.g., Amalfitano etal., 1998 J. Virol. 72:926-933; Hodges, et al., 2000 J Gene Med2:250-259).

As would be recognized by the skilled artisan, the adenovirus vectorsfor use in the present invention can be successfully grown to hightiters using an appropriate packaging cell line that constitutivelyexpresses E2b gene products and products of any of the necessary genesthat may have been deleted. In certain embodiments, HEK-293-derivedcells that not only constitutively express the E1 and DNA polymeraseproteins, but also the Ad-preterminal protein, can be used. In oneembodiment, E.C7 cells are used to successfully grow high titer stocksof the adenovirus vectors (see e.g., Amalfitano et al., J. Virol. 199872:926-933; Hodges, et al. J Gene Med 2/250-259 (2000)).

In order to delete critical genes from self-propagating adenovirusvectors, the proteins encoded by the targeted genes can first becoexpressed in HEK-293 cells, or similar, along with the E1 proteins.For example, only those proteins which are non-toxic when coexpressedconstitutively (or toxic proteins inducibly-expressed) can beselectively utilized. Coexpression in HEK-293 cells of the E1 and E4genes is possible (for example utilizing inducible, not constitutive,promoters) according to the methods in Yeh et al. (1996) J. Virol.70:559; Wang et al. (1995) Gene Therapy 2:775; and Gorziglia et al.(1996) J. Virol. 70:4173. The E1 and protein IX genes, a virionstructural protein, can be coexpressed [Caravokyri and Leppard (1995) J.Virol. 69:6627. Further coexpression of the E1, E4, and protein IX genesis also possible as described in Krougliak and Graham (1995) Hum. GeneTher. 6:1575. The E1 and 100 k genes can be successfully expressed intranscomplementing cell lines, as can E1 and protease genes (Oualikene,et al. Hum Gene Ther 11/1341-1353 (2000); Hodges, et al. J. Virol75/5913-5920 (2001)).

Cell lines coexpressing E1 and E2b gene products for use in growing hightiters of E2b deleted Ad particles are described in U.S. Pat. No.6,063,622. The E2b region encodes viral replication proteins, which areessential for Ad genome replication [Doerfler, supra and Pronk et al.(1992) Chromosoma 102:S39-545]. Useful cell lines constitutively expressthe approximately 140 kD Ad-DNA polymerase and/or the approximately 90kD preterminal protein. In particular, cell lines that have high-level,constitutive coexpression of the E1, DNA polymerase, and preterminalproteins, without toxicity (e.g. E.C7), are desirable for use inpropagating Ad for use in multiple vaccinations. These cell lines permitthe propagation of adenovirus vectors deleted for the E1, DNApolymerase, and preterminal proteins.

The recombinant Ad of the present invention can be propagated usingtechniques known in the art. For example, tissue culture platescontaining E.C7 cells can be infected with the adenovirus vector virusstocks at an appropriate MOI (e.g., 5) and incubated at 37.0.degree. C.for 40-96 h. The infected cells can be harvested, resuspended in 10 mMTris-Cl (pH 8.0), and sonicated, and the virus is purified by two roundsof cesium chloride density centrifugation. In certain techniques, thevirus containing band is desalted over a Sephadex CL-6B column(Pharmacia Biotech, Piscataway, N.J.), sucrose or glycerol is added, andaliquots are stored at −80.degree. C. In some embodiments, the virus isplaced in a solution designed to enhance its stability, such as A195(Evans, et al. J Pharm Sci 93/2458-2475 (2004)). The titer of the stockcan be measured (e.g., by measurement of the optical density at 260 nmof an aliquot of the virus after SDS lysis). In another embodiment,plasmid DNA, either linear or circular, encompassing the entirerecombinant E2b deleted adenovirus vector is transfected into E.C7, orsimilar cells, and incubated at 37.0.degree. C. until evidence of viralproduction is present (e.g. the cytopathic effect). The conditionedmedia from these cells can be used to infect more E.C7, or similarcells, to expand the amount of virus produced, before purification.Purification can be accomplished, for example, by two rounds of cesiumchloride density centrifugation or selective filtration. In certainembodiments, the virus may be purified by column chromatography, usingcommercially available products (e.g. Adenopure from Puresyn, Inc.,Malvern, Pa.) or custom made chromatographic columns.

Generally, the compositions of the present invention comprises enough ofthe virus to ensure that the cells to be infected are confronted with acertain number of viruses. Thus, in various embodiments, the presentinvention provides a stock of recombinant Ad, preferably an RCA-freestock of recombinant Ad. The preparation and analysis of Ad stocks iswell known in the art. Viral stocks can vary considerably in titer,depending largely on viral genotype and the protocol and cell lines usedto prepare them. The viral stocks of the present invention can have atiter of at least about 10⁶, 10⁷, or 10⁸ pfu/ml, and many such stockscan have higher titers, such as at least about 10⁹, 10¹⁰, 10¹¹, or 10¹²pfu/ml. Depending on the nature of the recombinant virus and thepackaging cell line, it is possible that a viral stock of the presentinvention can have a titer of even about 10¹³ particles/ml or higher.

Further information on viral delivery systems is known in the art andcan be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci.USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103,1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112,4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627,1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc.Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl.Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993,which are each herein incorporated by reference in their entirety.

Heterologous Nucleic Acid

The adenovirus vectors of the present invention typically compriseheterologous nucleic acid sequences that encode one or more targetantigens of interest, or variants, fragments or fusions thereof, againstwhich it is desired to generate an immune response. In some embodiments,the adenovirus vectors of the present invention comprise heterologousnucleic acid sequences that encode one or more proteins, variantsthereof, fusions thereof, or fragments thereof, that can modulate theimmune response. In a further embodiment of the invention, theadenovirus vector of the present invention encodes one or moreantibodies against specific antigens, such as anthrax protectiveantigen, permitting passive immunotherapy. In certain embodiments, theadenovirus vectors of the present invention comprise heterologousnucleic acid sequences encoding one or more proteins having therapeuticeffect (e.g., anti-viral, anti-bacterial, anti-parasitic, or anti-tumorfunction). Thus the present invention provides the Second Generation E2bdeleted adenovirus vectors that comprise a heterologous nucleic acidsequence. In some embodiments, the heterologous nucleic acid sequence iscolorectal embryonic antigen (CEA) or a variant, a part, or a variantpart thereof.

As such, the present invention further provides nucleic acid sequences,also referred to herein as polynucleotides, that encode one or moretarget antigens of interest, or fragments or variants thereof. As such,the present invention provides polynucleotides that encode targetantigens from any source as described further herein, vectors comprisingsuch polynucleotides and host cells transformed or transfected with suchexpression vectors. The terms “nucleic acid” and “polynucleotide” areused essentially interchangeably herein. As will be also recognized bythe skilled artisan, polynucleotides of the invention may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(e.g. genomic, cDNA, or synthetic) or RNA molecules. RNA molecules mayinclude HnRNA molecules, which contain introns and correspond to a DNAmolecule in a one-to-one manner, and mRNA molecules, which do notcontain introns. Additional coding or non-coding sequences may, but neednot, be present within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials. An isolated polynucleotide, as used herein, meansthat a polynucleotide is substantially away from other coding sequences.For example, an isolated DNA molecule as used herein does not containlarge portions of unrelated coding DNA, such as large chromosomalfragments or other functional genes or polypeptide coding regions. Thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment recombinantly in thelaboratory.

As will be understood by those skilled in the art, the polynucleotidesof this invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express target antigens as describedherein, fragments of antigens, peptides and the like. Such segments maybe naturally isolated, or modified synthetically by the hand of man.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a target antigen polypeptide/protein/epitope ofthe invention or a portion thereof) or may comprise a sequence thatencodes a variant, fragment, or derivative of such a sequence. Incertain embodiments, the polynucleotide sequences set forth hereinencode target antigen proteins as described herein. In some embodiments,polynucleotides represent a novel gene sequence that has been optimizedfor expression in specific cell types (i.e. human cell lines) that maysubstantially vary from the native nucleotide sequence or variant butencode a similar protein antigen.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to native sequencesencoding proteins (e.g., target antigens of interest) as describedherein, for example those comprising at least 70% sequence identity,preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a native polynucleotide sequenceencoding the polypeptides of this invention using the methods describedherein, (e.g., BLAST analysis using standard parameters, as describedbelow). One skilled in this art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. In some embodiments, the present invention providespolynucleotides encoding a protein comprising for example at least 70%sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% or higher, sequence identity compared to a proteinsequence encoded by a native polynucleotide sequence of this inventionusing the methods described herein.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the epitope of the polypeptide encoded by thevariant polynucleotide or such that the immunogenicity of theheterologous target protein is not substantially diminished relative toa polypeptide encoded by the native polynucleotide sequence. In somecases, said one or more substitutions, additions, deletions and/orinsertions may result in an increased immunogenicity of the epitope ofthe polypeptide encoded by the variant polynucleotide. As describedelsewhere herein, the polynucleotide variants preferably encode avariant of the target antigen, or a fragment (e.g., an epitope) thereofwherein the propensity of the variant polypeptide or fragment (e.g.,epitope) thereof to react with antigen-specific antisera and/or T-celllines or clones is not substantially diminished, but optionallysubstantially increased, relative to the native polypeptide. The term“variants” should also be understood to encompass homologous genes ofxenogenic origin. In particular embodiments, variants or fragments oftarget antigens are modified such that they have one or more reducedbiological activities. For example, an oncogenic protein target antigenmay be modified to reduce or eliminate the oncogenic activity of theprotein, or a viral protein may be modified to reduce or eliminate oneor more activities or the viral protein. An example of a modified CEAprotein is a CEA having a N610D mutation, resulting in a variant proteinwith increased immunogenicity.

The present invention provides polynucleotides that comprise or consistof at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 11, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or morecontiguous nucleotides encoding a polypeptide, including target proteinantigens, as described herein, as well as all intermediate lengths therebetween. It will be readily understood that “intermediate lengths”, inthis context, means any length between the quoted values, such as 16,17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53,etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including allintegers through 200-500; 500-1,000, and the like. A polynucleotidesequence as described herein may be extended at one or both ends byadditional nucleotides not found in the native sequence encoding apolypeptide as described herein, such as an epitope or heterologoustarget protein. This additional sequence may consist of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides ormore, at either end of the disclosed sequence or at both ends of thedisclosed sequence.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, expression controlsequences, polyadenylation signals, additional restriction enzyme sites,multiple cloning sites, other coding segments, and the like, such thattheir overall length may vary considerably. It is therefore contemplatedthat a nucleic acid fragment of almost any length may be employed, withthe total length preferably being limited by the ease of preparation anduse in the intended recombinant DNA protocol. For example, illustrativepolynucleotide segments with total lengths of about 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10,000, about 500, about 200, about100, about 50 base pairs in length, and the like, (including allintermediate lengths) are contemplated to be useful in manyimplementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Saitou, N. Nei, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nucl. Acids Res.25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 can be used, for example withthe parameters described herein, to determine percent sequence identityfor the polynucleotides of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. In one illustrative example, cumulativescores can be calculated using, for nucleotide sequences, the parametersM (reward score for a pair of matching residues; always >0) and N(penalty score for mismatching residues; always <0). Extension of theword hits in each direction are halted when: the cumulative alignmentscore falls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a particular antigen of interest, or fragmentthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention. Further, alleles ofthe genes comprising the polynucleotide sequences provided herein arewithin the scope of the present invention. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

In another embodiment of the invention, a mutagenesis approach, such assite-specific mutagenesis, is employed for the preparation of variantsand/or derivatives of the target antigen sequences, or fragmentsthereof, as described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provide astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of an epitope comprised in a polypeptide or theoncogenicity of a target antigen. Assays to test the immunogenicity of apolypeptide or variant thereof are well known in the art and include,but are not limited to, T cell cytotoxicity assays (CTL/chromium releaseassays), T cell proliferation assays, intracellular cytokine staining,ELISA, ELISpot, etc. The techniques of site-specific mutagenesis arewell known in the art, and are widely used to create variants of bothpolypeptides and polynucleotides. For example, site-specific mutagenesisis often used to alter a specific portion of a DNA molecule. In suchembodiments, a primer comprising typically about 14 to about 25nucleotides or so in length is employed, with about 5 to about 10residues on both sides of the junction of the sequence being altered.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982.

Polynucleotide segments or fragments encoding the polypeptides of thepresent invention may be readily prepared by, for example, directlysynthesizing the fragment by chemical means, as is commonly practicedusing an automated oligonucleotide synthesizer. Also, fragments may beobtained by application of nucleic acid reproduction technology, such asthe PCR.technology of U.S. Pat. No. 4,683,202, by introducing selectedsequences into recombinant vectors for recombinant production, and byother recombinant DNA techniques generally known to those of skill inthe art of molecular biology (see for example, Current Protocols inMolecular Biology, John Wiley and Sons, NY, N.Y.).

In order to express a desired target antigen polypeptide or fragment orvariant thereof, or fusion protein comprising any of the above, asdescribed herein, the nucleotide sequences encoding the polypeptide, orfunctional equivalents, are inserted into an appropriate Ad as describedelsewhere herein using recombinant techniques known in the art. Theappropriate adenovirus vector may contain the necessary elements for thetranscription and translation of the inserted coding sequence and anydesired linkers. Methods which are well known to those skilled in theart may be used to construct these adenovirus vectors containingsequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Amalfitano et al., 1998 J. Virol. 72:926-933; Hodges, etal., 2000 J Gene Med 2:250-259; Sambrook, J. et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in MolecularBiology, John Wiley & Sons, New York. N.Y.

A variety of vector/host systems may be utilized to contain and producepolynucleotide sequences. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA vectors; yeast transformed withyeast vectors; insect cell systems infected with virus vectors (e.g.,baculovirus); plant cell systems transformed with virus vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or withbacterial vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anadenovirus vector may include those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, sequences encoding apolypeptide of interest may be ligated into an Adtranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon can be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162). Specific terminationsequences, either for transcription or translation, may also beincorporated in order to achieve efficient translation of the sequenceencoding the polypeptide of choice.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products (e.g., target antigens of interest),using either polyclonal or monoclonal antibodies specific for theproduct are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson a given polypeptide may be preferred for some applications, but acompetitive binding assay may also be employed. These and other assaysare described, among other places, in Hampton, R. et al. (1990;Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) andMaddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

The adenovirus vectors of the present invention comprise nucleic acidsequences encoding one or more antigens of interest, or variants orfragments thereof. The nucleic acid sequence may also contain a productthat can be detected or selected for. As referred to herein, a“reporter” gene is one whose product can be detected, such as byfluorescence, enzyme activity on a chromogenic or fluorescent substrate,and the like or selected for by growth conditions.

Such reporter genes include, without limitation, green fluorescentprotein (GFP), β-galactosidase, chloramphenicol acetyltransferase (CAT),luciferase, neomycin phosphotransferase, secreted alkaline phosphatase(SEAP), and human growth hormone (HGH). Selectable markers include drugresistances, such as neomycin (G418), hygromycin, and the like.

The nucleic acid encoding an antigen of interest may also comprise apromoter or expression control sequence. This is a nucleic acid sequencethat controls expression of the nucleic acid sequence encoding a targetantigen and generally is active or activatable in the targeted cell. Thechoice of the promoter will depend in part upon the targeted cell typeand the degree or type of control desired. Promoters that are suitablewithin the context of the present invention include, without limitation,constitutive, inducible, tissue specific, cell type specific, temporalspecific, or event-specific.

Examples of constitutive or nonspecific promoters include the SV40 earlypromoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat.No. 5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062),bovine papilloma virus promoter, and adenovirus promoter. In addition toviral promoters, cellular promoters are also amenable within the contextof this invention. In particular, cellular promoters for the so-calledhousekeeping genes are useful (e.g., f3-actin). Viral promoters aregenerally stronger promoters than cellular promoters.

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone, metallothionein,inducible by heavy metals, and promoters with cAMP response elements,inducible by cAMP, heat shock, promoter. By using an inducible promoter,the nucleic acid may be delivered to a cell and will remain quiescentuntil the addition of the inducer. This allows further control on thetiming of production of the protein of interest.

Event-type specific promoters are active or upregulated only upon theoccurrence of an event, such as tumorigenicity or viral infection, forexample. The HIV LTR is a well-known example of an event-specificpromoter. The promoter is inactive unless the tat gene product ispresent, which occurs upon viral infection. Some event-type promotersare also tissue-specific. Preferred event-type specific promotersinclude promoters activated upon viral infection.

Examples of promoters discussed herein include, but are not limited to,promoters for alphafetoprotein, alpha actin, myo D, carcinoembryonicantigen, VEGF-receptor (GenBank Accession No. X89776); FGF receptor; TEKor tie 2 (GenBank Accession No. L06139); tie (GenBank Accession Nos.X60954; S89716); urokinase receptor (GenBank Accession No. S78532); E-and P-selectins (GenBank Accession Nos. M64485; L01874); VCAM-1 (GenBankAccession No. M92431); endoglin (GenBank Accession No. HSENDOG);endosialin (Rettig et al., PNAS 89:10832, 1992); alpha V-beta3 integrin(Villa-Garcia et al., Blood 3:668, 1994; Donahue et al., BBA 1219:228,1994); endothelin-1 (GenBank Accession Nos. M25377; J04819; J05489);ICAM-3 (GenBank Accession No. S50015); E9 antigen (Wang et al., Int. J.Cancer 54:363, 1993); von Willebrand factor (GenBank Accession Nos.HUMVWFI; HUMVWFA); CD44 (GenBank Accession No. HUMCD44B); CD40 (GenBankAccession Nos. HACD40L; HSCD405FR); vascular-endothelial cadherin(Martin-Padura et al., J. Pathol. 175:51, 1995); notch 4 (Uyttendaele etal., Development 122:2251, 1996) high molecular weightmelanoma-associated antigen; prostate specific antigen-1, probasin, FGFreceptor, VEGF receptor, erb B2; erb B3; erb B4; MUC-1; HSP-27; int-1;int-2, CEA, HBEGF receptor; EGF receptor; tyrosinase, MAGE, IL-2receptor; prostatic acid phosphatase, probasin, prostate specificmembrane antigen, alpha-crystallin, PDGF receptor, integrin receptor,α-actin, SM1 and SM2 myosin heavy chains, calponin-hl, SM22 alphaangiotensin receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, immunoglobulin heavy chain,immunoglobulin light chain, CD4, and the like are useful within thecontext of this invention.

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of the polynucleotide. Multiple repressor elements may beinserted in the promoter region. Repression of transcription isindependent of the orientation of repressor elements or distance fromthe promoter. One type of repressor sequence is an insulator sequence.Such sequences inhibit transcription (Dunaway et al., Mol Cell Biol 17:182-9, 1997; Gdula et al., Proc Natl Acad Sci USA 93:9378-83, 1996, Chanet al., J Virol 70: 5312-28, 1996; Scott and Geyer, EMBO J. 14: 6258-67,1995; Kalos and Fournier, Mol Cell Biol 15: 198-207, 1995; Chung et al.,Cell 74: 505-14, 1993) and can silence background transcription.

Negative regulatory elements can be located in the promoter regions of anumber of different genes. The repressor element can function as arepressor of transcription in the absence of factors, such as steroids,as does the NSE in the promoter region of the ovalbumin gene (Haecker etal., Mol. Endocrinology. 9:1113-1126, 1995). These negative regulatoryelements can bind specific protein complexes from oviduct, none of whichare sensitive to steroids. Three different elements are located in thepromoter of the ovalbumin gene. Oligonucleotides corresponding toportions of these elements can repress viral transcription of the TKreporter. One of the silencer elements shares sequence identity withsilencers in other genes (TCTCTCCNA (SEQ ID NO: 6)).

Further, repressor elements can be located in the promoter region of avariety of genes, including the collagen II gene, for example. Nuclearfactors from HeLa cells can bind specifically to DNA fragmentscontaining the silencer region (Savanger et al., J. Biol. Chem.265(12):6669-6674, 1990). Repressor elements may play a role regulatingtranscription in the carbamyl phosphate synthetase gene (Goping et al.,Nucleic Acid Research 23(10):1717-1721, 1995). This gene is expressed inonly two different cell types, hepatocytes and epithelial cells of theintestinal mucosa. Negative regulatory regions are also found in thepromoter region of the choline acetyltransferase gene, the albuminpromoter (Hu et al., J. Cell Growth Differ. 3(9):577-588, 1992),phosphoglycerate kinase (PGK-2) gene promoter (Misuno et al., Gene119(2):293-297, 1992), and in the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene, in which the negative regulatory element inhibitstranscription in non-hepatic cell lines (Lemaigre et al., Mol. Cell.Biol. 11(2):1099-1106). Furthermore, the negative regulatory elementTse-1 is located in a number of liver specific genes, including tyrosineaminotransferase (TAT). TAT gene expression is liver specific andinducible by both glucocorticoids and the cAMP signaling pathway. ThecAMP response element (CRE) can ask as the target for repression byTse-1 and hepatocyte-specific elements (Boshart et al., Cell61(5):905-916, 1990). Accordingly, it is clear that varieties of suchelements are known or are readily identified.

In certain embodiments, elements that increase the expression of thedesired target antigen are incorporated into the nucleic acid sequenceof the adenovirus vectors described herein. Such elements include, butare not limited to internal ribosome binding sites (IRES; Wang andSiddiqui, Curr. Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld andSemler, Curr. Top. Microbiol. Immunol. 203:65, 1995; Rees et al.,Biotechniques 20:102, 1996; Sugimoto et al., Biotechnology 12:694,1994). IRES can increase translation efficiency. As well, othersequences may enhance expression. For some genes, sequences especiallyat the 5′ end may inhibit transcription and/or translation. Thesesequences are usually palindromes that can form hairpin structures. Insome cases, such sequences in the nucleic acid to be delivered aredeleted. Expression levels of the transcript or translated product canbe assayed to confirm or ascertain which sequences affect expression.Transcript levels may be assayed by any known method, including Northernblot hybridization, RNase probe protection and the like. Protein levelsmay be assayed by any known method, including ELISA.

As would be recognized by the skilled artisan, the adenovirus vectors ofthe present invention comprising heterologous nucleic acid sequences canbe generated using recombinant techniques known in the art, such asthose described in Maione et al., 2001 Proc Natl Acad Sci USA,98:5986-5991; Maione et al., 2000 Hum Gene Ther 11:859-868; Sandig etal. 2000 Proc Natl Acad Sci USA, 97:1002-1007; Harui et al. 2004 GeneTherapy, 11:1617-1626; Parks et al., 1996 Proc Natl Acad Sci USA,93:13565-13570; DelloRusso et al., 2002 Proc Natl Acad Sci USA,99:12979-12984; Current Protocols in Molecular Biology, John Wiley andSons, NY, N.Y.).

As noted above, the adenovirus vectors of the present invention comprisenucleic acid sequences that encode one or more target proteins orantigens of interest. In this regard, the vectors may contain nucleicacid encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more different target antigens of interest. The targetantigens may be a full length protein or may be a fragment (e.g., anepitope) thereof. The adenovirus vectors may contain nucleic acidsequences encoding multiple fragments or epitopes from one targetprotein of interest or may contain one or more fragments or epitopesfrom numerous different target proteins of interest. In someembodiments, the target antigen or protein comprises CEA.

The term “target antigen” or “target protein” as used herein refers to amolecule, such as a protein, against which an immune response is to bedirected. The target antigen may comprise any substance against which itis desirable to generate an immune response but generally, the targetantigen is a protein. A target antigen may comprise a full lengthprotein or a fragment thereof that induces an immune response (i.e., animmunogenic fragment). A target antigen or fragment thereof may bemodified, e.g., to reduce one or more biological activities of thetarget antigen or to enhance its immunogenicity. In some embodiments,the target antigen or target protein is CEA.

An “immunogenic fragment”, as used herein is a fragment of a polypeptidethat is specifically recognized (i.e., specifically bound) by a B-celland/or T-cell surface antigen receptor resulting in the generation of animmune response specifically against the fragment. In certainembodiments, immunogenic fragments bind to an MHC class I or class IImolecule. As used herein, an immunogenic fragment is said to “bind to”an MHC class I or class II molecule if such binding is detectable usingany assay known in the art. For example, the ability of a polypeptide tobind to MHC class I may be evaluated indirectly by monitoring theability to promote incorporation of ¹²⁵I labeled β.2-microglobulin (β 2m) into MHC class I/β2 m/peptide heterotrimeric complexes (see Parker etal., J. Immunol. 152:163, 1994). Alternatively, functional peptidecompetition assays that are known in the art may be employed.Immunogenic fragments of polypeptides may generally be identified usingwell known techniques, such as those summarized in Paul, FundamentalImmunology, 3rd ed., 243-247 (Raven Press, 1993) and references citedtherein. Representative techniques for identifying immunogenic fragmentsinclude screening polypeptides for the ability to react withantigen-specific antisera and/or T-cell lines or clones. An immunogenicfragment of a particular target polypeptide is a fragment that reactswith such antisera and/or T-cells at a level that is not substantiallyless than the reactivity of the full length target polypeptide (e.g., inan ELISA and/or T-cell reactivity assay). In other words, an immunogenicfragment may react within such assays at a level that is similar to orgreater than the reactivity of the full length polypeptide. Such screensmay generally be performed using methods well known to those of ordinaryskill in the art, such as those described in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Target antigens of the present invention include but are not limited toantigens derived from any of a variety of infectious agents or cancercells. An infectious agent may refer to any living organism capable ofinfecting a host. Cancer typically includes a neoplastic cell.Infectious agents include, for example, bacteria, any variety ofviruses, such as, single stranded RNA viruses, single stranded DNAviruses, fungi, parasites, and protozoa. Examples of infectious agentsinclude, but are not limited to, Actinobacillus spp., Actinomyces spp.,Adenovirus (types 1, 2, 3, 4, 5 et 7), Adenovirus (types 40 and 41),Aerococcus spp., Aeromonas hydrophila, Ancylostoma duodenale,Angiostrongylus cantonensis, Ascaris lumbricoides, Ascaris spp.,Aspergillus spp., Babesia spp, B. microti, Bacillus anthracis, Bacilluscereus, Bacteroides spp., Balantidium coli, Bartonella bacilliformis,Blastomyces dermatitidis, Bluetongue virus, Bordetella bronchiseptica,Bordetella pertussis, Borrelia afzelii, Borrelia burgdorferi, Borreliagarinii, Branhamella catarrhalis, Brucella spp. (B. abortus, B. canis,B. melitensis, B. suis), Brugia spp., Burkholderia, (Pseudomonas)mallei, Burkholderia (Pseudomonas) pseudomallei, California serogroup,Campylobacter fetus subsp. Fetus, Campylobacter jejuni, C. coli, C.fetus subsp. Jejuni, Candida albicans, Capnocytophaga spp., Chikungunyavirus, Chlamydia psittaci, Chlamydia trachomatis, Citrobacter spp.,Clonorchis sinensis, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Clostridium spp. (with theexception of those species listed above), Coccidioides immitis, Coloradotick fever virus, Corynebacterium diphtheriae, Coxiella burnetii,Coxsackievirus, Creutzfeldt-Jakob agent, Kuru agent, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidiumparvum, Cytomegalovirus, Cyclospora cayatanesis, Dengue virus (1, 2, 3,4), Diphtheroids, Eastern (Western) equine encephalitis virus, Ebolavirus, Echinococcus granulosus, Echinococcus multilocularis, Echovirus,Edwardsiella tarda, Entamoeba histolytica, Enterobacter spp.,Enterovirus 70, Epidermophyton floccosum, Ehrlichia spp, Ehrlichiasennetsu, Microsporum spp. Trichophyton spp., Epstein-Barr virus,Escherichia coli, enterohemorrhagic, Escherichia coli, enteroinvasive,Escherichia coli, enteropathogenic, Escherichia coli, enterotoxigenic,Fasciola hepatica, Francisella tularensis, Fusobacterium spp., Gemellahaemolysans, Giardia lamblia, Guanarito virus, Haemophilus ducreyi,Haemophilus influenzae (group b), Hantavirus, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis Evirus, Herpes simplex virus, Herpesvirus simiae, Histoplasma capsulatum,Human coronavirus, Human immunodeficiency virus, Human papillomavirus,Human rotavirus, Human T-lymphotrophic virus, Influenza virus includingH5N1, Junin virus/Machupo virus, Klebsiella spp., Kyasanur Forestdisease virus, Lactobacillus spp., Lassa virus, Legionella pneumophila,Leishmania major, Leishmania infantum, Leishmania spp., Leptospirainterrogans, Listeria monocytogenes, Lymphocytic choriomeningitis virus,Machupo virus, Marburg virus, Measles virus, Micrococcus spp., Moraxellaspp., Mycobacterium spp. (other than M. bovis, M. tuberculosis, M.avium, M. leprae), Mycobacterium tuberculosis, M. bovis, Mycoplasmahominis, M. orale, M. salivarium, M. fermentans, Mycoplasma pneumoniae,Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseriameningitides, Neisseria spp. (other than N. gonorrhoeae and N.meningitidis), Nocardia spp., Norwalk virus, Omsk hemorrhagic fevervirus, Onchocerca volvulus, Opisthorchis spp., Parvovirus B19,Pasteurella spp., Peptococcus spp., Peptostreptococcus spp., Plasmodiumfalciparum, Plasmodium vivax, Plasmodium spp., Plesiomonas shigelloides,Powassan encephalitis virus, Proteus spp., Pseudomonas spp. (other thanP. mallei, P. pseudomallei), Rabies virus, Respiratory syncytial virus,Rhinovirus, Rickettsia akari, Rickettsia prowazekii, R. Canada,Rickettsia rickettsii, Rift Valley virus, Ross river virus/O'Nyong-Nyongvirus, Rubella virus, Salmonella choleraesuis, Salmonella paratyphi,Salmonella typhi, Salmonella spp. (with the exception of those specieslisted above), Schistosoma spp., Scrapie agent, Serratia spp., Shigellaspp., Sindbis virus, Sporothrix schenckii, St. Louis encephalitis virus,Murray Valley encephalitis virus, Staphylococcus aureus, Streptobacillusmoniliformis, Streptococcus agalactiae, Streptococcus faecalis,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcussalivarius, Taenia saginata, Taenia solium, Toxocara canis, T. cati, T.cruzi, Toxoplasma gondii, Treponema pallidum, Trichinella spp.,Trichomonas vaginalis, Trichuris trichiura, Trypanosoma brucei,Trypanosoma cruzi, Ureaplasma urealyticum, Vaccinia virus,Varicella-zoster virus, eastern equine encephalitis virus (EEEV), severeacute respiratory virus (SARS), Venezuelan equine encephalitis virus(VEEV), Vesicular stomatitis virus, Vibrio cholerae, serovar 01, Vibrioparahaemolyticus, West Nile virus, Wuchereria bancrofti, Yellow fevervirus, Yersinia enterocolitica, Yersinia pseudotuberculosis, andYersinia pestis.

Examples of infectious agents associated with human malignancies includeEpstein-Barr virus, Helicobacter pylori, Hepatitis B virus, Hepatitis Cvirus, Human heresvirus-8, Human immunodeficiency virus, Humanpapillomavirus, Human T cell leukemia virus, liver flukes, andSchistosoma haematobium.

A number of viruses are associated with viral hemorrhagic fever,including filoviruses (e.g., Ebola, Marburg, and Reston), arenaviruses(e.g. Lassa, Junin, and Machupo), and bunyaviruses. In addition,phleboviruses, including, for example, Rift Valley fever virus, havebeen identified as etiologic agents of viral hemorrhagic fever.Etiological agents of hemorrhagic fever and associated inflammation mayalso include paramyxoviruses, particularly respiratory syncytial virus(Feldmann, H. et al. (1993) Arch Virol Suppl. 7:81-100). In addition,other viruses causing hemorrhagic fevers in man have been identified asbelonging to the following virus groups: togavirus (Chikungunya),flavivirus (dengue, yellow fever, Kyasanur Forest disease, Omskhemorrhagic fever), nairovirus (Crimian-Congo hemorrhagic fever) andhantavirus (hemorrhagic fever with renal syndrome, nephropathicepidemia). Furthermore, Sin Nombre virus was identified as the etiologicagent of the 1993 outbreak of hantavirus pulmonary syndrome in theAmerican Southwest.

Target antigens may include proteins, or variants or fragments thereof,produced by any of the infectious organisms described herein, such as,but not limited to, viral coat proteins, i.e., influenza neuraminidaseand hemagglutinin, HIV gp160 or derivatives thereof, HIV Gag, HIV Nef,HIV Pol, SARS coat proteins, herpes virion proteins, WNV proteins, etc.Target antigens may also include bacterial surface proteins includingpneumococcal PsaA, PspA, LytA, surface or virulence associated proteinsof bacterial pathogens such as Nisseria gonnorhea, outer membraneproteins or surface proteases.

Target antigens may also include proteins, or variants or fragmentsthereof, of infectious agents associated with human malignancies such asthe human papillomavirus (HPV) oncoproteins E6 and E7. In certainembodiments, the oncoprotein may be modified to produce a non-oncogenicvariant or a variant having reduced oncogenicity relative to the wildtype protein. For example, the portion of the peptide that isresponsible for binding a tumor suppressor protein (e.g., p53 and pRb)may be deleted or modified so that it no longer interacts with the tumorsuppressor protein. As another example, an oncoprotein that is a kinase,such as Her2/neu, may be kinase-inactivated, e.g., by point mutation. Insome instances, two or more target antigens may be used duringimmunization. For example, the E6 and E7 antigens can be combined in afusion protein, or separate vectors containing heterologous nucleotidesencoding the modified or unmodified E6 and E7 target antigens are usedin combination. For example, an Ad5-E6 vector can be administered withan Ad5-E7 vector. In this example, the Ad5-E6 vector and Ad5-E7 vectormay be administered simultaneously or they may be administeredsequentially.

Target antigens of the present invention include but are not limited toantigens derived from a variety of tumor proteins. Illustrative tumorproteins useful in the present invention include, but are not limited toany one or more of, WT1, HPV E6, HPV E7, p53, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, -10, GAGE-1, -2, -8,GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-1, MART-1, MC1R, Gp100, PSA,PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, Her2/neu,BRCA1, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2, SART-1,SART-3, WT1, AFP, β-catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V,G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE,SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML,LDLR/FUT, Pml/RARα, and TEL/AML1. These and other tumor proteins areknown to the skilled artisan. In some embodiments, parts or variants oftumor proteins are employed as target antigens. In some embodiments,parts or variants of tumor proteins being employed as target antigenshave a modified, for example, increased ability to effect and immuneresponse against the tumor protein or cells containing the same.

In some embodiments, a replication defective adenovirus vector, e.g. thevector identified by SEQ. ID. NO.:3, comprises a sequence encoding atarget antigen described herein, or a fragment, a variant, or a variantfragment thereof, at a suitable position on the sequence, for example ata position replacing the nucleic acid sequence encoding a CEA or avariant CEA, e.g. the sequence identified by SEQ. ID. NO.:1.

In certain embodiments tumor antigens may be identified directly from anindividual with cancer. In this regard, screens can be carried out usinga variety of known technologies. For example, in one embodiment, a tumorbiopsy is taken from a patient, RNA is isolated from the tumor cells andscreened using a gene chip (for example, from Affymetrix, Santa Clara,Calif.) and a tumor antigen is identified. Once the tumor target antigenis identified, it may then be cloned, expressed and purified usingtechniques known in the art. This target molecule is then linked to oneor more epitopes/cassettes of the present invention as described hereinand administered to the cancer patient in order to alter the immuneresponse to the target molecule isolated from the tumor. In this manner,“personalized vaccines” are contemplated within the context of theinvention. In certain embodiments, cancers may include carcinomas orsarcomas. In some embodiments, a personalized tumor antigen related toCEA is characterized from a patient and further utilized as the targetantigen as a whole, in part or as a variant.

The adenovirus vectors of the present invention may also include nucleicacid sequences that encode proteins that increase the immunogenicity ofthe target antigen. In this regard, the protein produced followingimmunization with the adenovirus vector containing such a protein may bea fusion protein comprising the target antigen of interest fused to aprotein that increases the immunogenicity of the target antigen ofinterest.

In one embodiment, such an “immunological fusion partner” is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences are described in U.S. PatentApplication 60/158,585 and U.S. Pat. No. 7,009,042, which are hereinincorporated by reference in their entirety. Briefly, Ra12 refers to apolynucleotide region that is a subsequence of a Mycobacteriumtuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KDmolecular weight encoded by a gene in virulent and avirulent strains ofM. tuberculosis. The nucleotide sequence and amino acid sequence ofMTB32A have been described (for example, U.S. Patent Application60/158,585; see also, Skeiky et al., Infection and Immun. 67:3998-4007(1999), incorporated herein by reference in their entirety). C-terminalfragments of the MTB32A coding sequence express at high levels andremain as soluble polypeptides throughout the purification process.Moreover, Ra12 may enhance the immunogenicity of heterologousimmunogenic polypeptides with which it is fused. One Ra12 fusionpolypeptide comprises a 14 KD C-terminal fragment corresponding to aminoacid residues 192 to 323 of MTB32A. Other Ra12 polynucleotides generallycomprise at least about 15 consecutive nucleotides, at least about 30nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

Within another embodiment, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). In some cases, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids). A protein D derivative may belipidated. Within certain embodiments, the first 109 residues of aLipoprotein D fusion partner is included on the N-terminus to providethe polypeptide with additional exogenous T-cell epitopes, which mayincrease the expression level in E. coli and may function as anexpression enhancer. The lipid tail may ensure optimal presentation ofthe antigen to antigen presenting cells. Other fusion partners includethe non-structural protein from influenzae virus, NS 1 (hemagglutinin)Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within anotherembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion can, for example, be found in theC-terminal region starting at residue 178. One particular repeat portionincorporates residues 188-305.

Methods of Use

The adenovirus vectors of the present invention can be used in a numberof vaccine settings for generating an immune response against one ormore target antigens as described herein. The adenovirus vectors are ofparticular importance because of the unexpected finding that they can beused to generate immune responses in subjects who have preexistingimmunity to Ad and can be used in vaccination regimens that includemultiple rounds of immunization using the adenovirus vectors, regimensnot possible using previous generation adenovirus vectors.

Generally, generating an immune response comprises an induction of ahumoral response and/or a cell-mediated response. In certainembodiments, it is desirable to increase an immune response against atarget antigen of interest. In certain circumstances, generating animmune response may involve a decrease in the activity and/or number ofcertain cells of the immune system or a decrease in the level and/oractivity of certain cytokines or other effector molecules. As such“generating an immune response” or “inducing an immune response”comprises any statistically significant change, e.g. increase ordecrease, in the number of one or more immune cells (T cells, B cells,antigen-presenting cells, dendritic cells, neutrophils, and the like) orin the activity of one or more of these immune cells (CTL activity, HTLactivity, cytokine secretion, change in profile of cytokine secretion,etc.).

The skilled artisan would readily appreciate that a number of methodsfor establishing whether an alteration in the immune response has takenplace are available. A variety of methods for detecting alterations inan immune response (e.g. cell numbers, cytokine expression, cellactivity) are known in the art and are useful in the context of theinstant invention. Illustrative methods are described in CurrentProtocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek,David H. Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley &Sons, NY, N.Y.) Ausubel et al. (2001 Current Protocols in MolecularBiology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.);Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring HarborLaboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.) and elsewhere.Illustrative methods useful in this context include intracellularcytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cellassays including chromium release or equivalent assays, and geneexpression analysis using any number of polymerase chain reaction (PCR)or RT-PCR based assays.

In certain embodiments, generating an immune response comprises anincrease in target antigen-specific CTL activity of between 1.5 and 5fold in a subject administered the adenovirus vectors of the inventionas compared to a control. In another embodiment, generating an immuneresponse comprises an increase in target-specific CTL activity of about2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in asubject administered the adenovirus vectors as compared to a control.

In a further embodiment, generating an immune response comprises anincrease in target antigen-specific HTL activity, such as proliferationof helper T cells, of between 1.5 and 5 fold in a subject administeredthe adenovirus vectors of the invention that comprise nucleic acidencoding the target antigen as compared to an appropriate control. Inanother embodiment, generating an immune response comprises an increasein target-specific HTL activity of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16,17, 18, 19, 20, or more fold as compared to a control. In this context,HTL activity may comprise an increase as described above, or decrease,in production of a particular cytokine, such as interferon-gamma(IFN-γ), interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-7, IL-12, IL-15,tumor necrosis factor-alpha (TNF-α), granulocyte macrophagecolony-stimulating factor (GM-CSF), granulocyte-colony stimulatingfactor (G-CSF), or other cytokine. In this regard, generating an immuneresponse may comprise a shift from a Th2 type response to a Th1 typeresponse or in certain embodiments a shift from a Th1 type response to aTh2 type response. In other embodiments, generating an immune responsemay comprise the stimulation of a predominantly Th1 or a Th2 typeresponse.

In a further embodiment, generating an immune response comprises anincrease in target-specific antibody production of between 1.5 and 5fold in a subject administered the adenovirus vectors of the presentinvention as compared to an appropriate control. In another embodiment,generating an immune response comprises an increase in target-specificantibody production of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19,20, or more fold in a subject administered the adenovirus vector ascompared to a control.

Thus the present invention provides methods for generating an immuneresponse against a target antigen of interest comprising administeringto the individual an adenovirus vector comprising: a) a replicationdefective adenovirus vector, wherein the adenovirus vector has adeletion in the E2b region, and b) a nucleic acid encoding the targetantigen; and readministering the adenovirus vector at least once to theindividual; thereby generating an immune response against the targetantigen. In certain embodiments, the present invention provides methodswherein the vector administered is not a gutted vector. In particularembodiments, the target antigen may be a wild-type protein, a fragment,a variant, or a variant fragment thereof. In some embodiments, thetarget antigen comprises CEA, a fragment, a variant, or a variantfragment thereof.

In a further embodiment, the present invention provides methods forgenerating an immune response against a target antigen in an individual,wherein the individual has preexisting immunity to Ad, by administeringto the individual an adenovirus vector comprising: a) a replicationdefective adenovirus vector, wherein the adenovirus vector has adeletion in the E2b region, and b) a nucleic acid encoding the targetantigen; and readministering the adenovirus vector at least once to theindividual; thereby generating an immune response against the targetantigen. In particular embodiments, the target antigen may be awild-type protein, a fragment, a variant, or a variant fragment thereof.In some embodiments, the target antigen comprises CEA, a fragment, avariant, or a variant fragment thereof.

With regard to preexisting immunity to Ad, this can be determined usingmethods known in the art, such as antibody-based assays to test for thepresence of Ad antibodies. Further, in certain embodiments, the methodsof the present invention include first determining that an individualhas preexisting immunity to Ad then administering the E2b deletedadenovirus vectors of the invention as described herein.

One embodiment of the invention provides a method of generating animmune response against one or more target antigens in an individualcomprising administering to the individual a first adenovirus vectorcomprising a replication defective adenovirus vector, wherein theadenovirus vector has a deletion in the E2b region, and a nucleic acidencoding at least one target antigen; administering to the individual asecond adenovirus vector comprising a replication defective adenovirusvector, wherein the adenovirus vector has a deletion in the E2b region,and a nucleic acid encoding at least one target antigen, wherein the atleast one target antigen of the second adenovirus vector is the same ordifferent from the at least one target antigen of the first adenovirusvector. In particular embodiments, the target antigen may be a wild-typeprotein, a fragment, a variant, or a variant fragment thereof. In someembodiments, the target antigen comprises CEA, a fragment, a variant, ora variant fragment thereof.

Thus, the present invention contemplates multiple immunizations with thesame E2b deleted adenovirus vector or multiple immunizations withdifferent E2b deleted adenovirus vectors. In each case, the adenovirusvectors may comprise nucleic acid sequences that encode one or moretarget antigens as described elsewhere herein. In certain embodiments,the methods comprise multiple immunizations with an E2b deletedadenovirus encoding one target antigen, and re-administration of thesame adenovirus vector multiple times, thereby inducing an immuneresponse against the target antigen. In some embodiments, the targetantigen comprises CEA, a fragment, a variant, or a variant fragmentthereof.

In a further embodiment, the methods comprise immunization with a firstadenovirus vector that encodes one or more target antigens, and thenadministration with a second adenovirus vector that encodes one or moretarget antigens that may be the same or different from those antigensencoded by the first adenovirus vector. In this regard, one of theencoded target antigens may be different or all of the encoded antigensmay be different, or some may be the same and some may be different.Further, in certain embodiments, the methods include administering thefirst adenovirus vector multiple times and administering the secondadenovirus multiple times. In this regard, the methods compriseadministering the first adenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or more times and administering the secondadenovirus vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore times. The order of administration may comprise administering thefirst adenovirus one or multiple times in a row followed byadministering the second adenovirus vector one or multiple times in arow. In certain embodiments, the methods include alternatingadministration of the first and the second adenovirus vectors as oneadministration each, two administrations each, three administrationseach, and so on. In certain embodiments, the first and the secondadenovirus vectors are administered simultaneously. In otherembodiments, the first and the second adenovirus vectors areadministered sequentially. In some embodiments, the target antigencomprises CEA, a fragment, a variant, or a variant fragment thereof.

As would be readily understood by the skilled artisan, more than twoadenovirus vectors may be used in the methods of the present invention.Three, 4, 5, 6, 7, 8, 9, 10 or more different adenovirus vectors may beused in the methods of the invention. In certain embodiments, themethods comprise administering more than one E2b deleted adenovirusvector at a time. In this regard, immune responses against multipletarget antigens of interest can be generated by administering multipledifferent adenovirus vectors simultaneously, each comprising nucleicacid sequences encoding one or more target antigens. In someembodiments, the one or more target antigen comprises CEA, a fragment, avariant, or a variant fragment thereof.

The present invention provides methods of generating an immune responseagainst any target antigen, such as those described elsewhere herein.

In certain embodiments, the adenovirus vectors are used to generate animmune response against a cancer. In this regard, the methods includegenerating an immune response against carcinomas or sarcomas such assolid tumors, lymphomas or leukemias. Thus, the adenovirus vectorsdescribed herein are used to generate an immune response against acancer including but not limited to carcinomas or sarcomas such asneurologic cancers, melanoma, non-Hodgkin's lymphoma, Hodgkin's disease,leukemias, plasmocytomas, adenomas, gliomas, thymomas, breast cancer,prostate cancer, colo-rectal cancer, kidney cancer, renal cellcarcinoma, uterine cancer, pancreatic cancer, esophageal cancer, lungcancer, ovarian cancer, cervical cancer, testicular cancer, gastriccancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),and chronic lymphocytic leukemia (CLL), or other cancers. In someembodiments, the target antigen comprises CEA, a fragment, a variant, ora variant fragment thereof.

Further, in this regard, the cancer target antigens may include but arenot limited to antigens derived from a variety of tumor proteins.Illustrative tumor proteins useful in the present invention include, butare not limited to any one or more of, p53, HPV E6, HPV E7, MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6,-10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-1, MART-1,MC1R, Gp100, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA,Cyp-B, Her2/neu, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2,SART-1, SART-3, WT1, AFP, β-catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M,GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m,RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl,ETV6/AML, LDLR/FUT, Pml/RARα, and TEL/AML1. These and other tumorproteins are known to the skilled artisan. In particular embodiments,the target antigen may be one of these and other tumor proteins, afragment, a variant, or a variant fragment thereof.

Methods are also provided for treating or ameliorating the symptoms ofany of the infectious diseases or cancers as described herein. Themethods of treatment comprise administering the adenovirus vectors oneor more times to individuals suffering from or at risk from sufferingfrom an infectious disease or cancer as described herein. As such, thepresent invention provides methods for vaccinating against infectiousdiseases or cancers in individuals who are at risk of developing such adisease. Individuals at risk may be individuals who may be exposed to aninfectious agent at some time or have been previously exposed but do notyet have symptoms of infection or individuals having a geneticpredisposition to developing a cancer or being particularly susceptibleto an infectious agent. Individuals suffering from an infectious diseaseor cancer described herein may be determined to express and/or present atarget antigen, which may be use to guide the therapies herein. Forexample, an example can be found to express and/or present a targetantigen and an adenovirus vector encoding the target antigen, a variant,a fragment or a variant fragment thereof may be administeredsubsequently.

The present invention contemplates the use of adenovirus vectors for thein vivo delivery of nucleic acids encoding a target antigen, or afragment, a variant, or a variant fragment thereof. Once injected into asubject, the nucleic acid sequence is expressed resulting in an immuneresponse against the antigen encoded by the sequence. The adenovirusvector vaccine is administered in an “effective amount”, that is, anamount of adenovirus vector that is effective in a selected route orroutes of administration to elicit an immune response as describedelsewhere herein. In certain embodiments, an effective amount is onethat induces an immune response effective to facilitate protection ortreatment of the host against the target infectious agent or cancer. Theamount of vector in each vaccine dose is selected as an amount whichinduces an immune, immunoprotective or other immunotherapeutic responsewithout significant adverse effects generally associated with typicalvaccines. Once vaccinated, subjects may be monitored to determine theefficacy of the vaccine treatment. Monitoring the efficacy ofvaccination may be performed by any method known to a person of ordinaryskill in the art. In some embodiments, blood or fluid samples may beassayed to detect levels of antibodies. In other embodiments, ELISpotassays may be performed to detect a cell-mediated immune response fromcirculating blood cells or from lymphoid tissue cells.

The adenovirus vectors of the invention are generally prepared as knownin the art (see e.g., Hodges et al., 2000 supra; or Amalfitano et al.,1998 supra). For example, in certain embodiments, tissue culture platescontaining E.C7 or C-7 cells are infected with the adenovirus vectorvirus stocks at an appropriate MOI (e.g., 5) and incubated at 37° C. for40 h. The infected cells are harvested, resuspended in an appropriatebuffer such as 10 mM Tris-Cl (pH 8.0), and sonicated, and the virus ispurified by two rounds of cesium chloride density centrifugation. Incertain techniques, the virus containing band is desalted over aSephadex CL-6B column (Pharmacia Biotech, Piscataway, N.J.), glycerol isadded to a concentration of 12%, and aliquots are stored at −80° C. Thetiter of the stock is measured (e.g. by measurement of the opticaldensity at 260 nm of an aliquot of the virus after SDS lysis). GMPprocedures for producing appropriate Ad stocks for human administrationare used where appropriate.

For administration, the adenovirus vector stock is combined with anappropriate buffer, physiologically acceptable carrier, excipient or thelike. In certain embodiments, an appropriate number of adenovirus vectorparticles are administered in an appropriate buffer, such as, sterilePBS. In certain circumstances it will be desirable to deliver theadenovirus vector compositions disclosed herein subcutaneously,parenterally, intravenously, intramuscularly, or even intraperitoneally.In certain embodiments, solutions of the active compounds as free baseor pharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. In other embodiments, E2b deleted adenovirusvectors may be delivered in pill form, delivered by swallowing or bysuppository.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions, for example, see U.S. Pat. No. 5,466,468, which is hereinincorporated by reference in its entirety. Fluid forms to the extentthat easy syringability exists may be preferred. Forms that are stableunder the conditions of manufacture and storage are within the bounds ofthis invention. In various embodiments, forms are preserved against thecontaminating action of microorganisms, such as bacteria, molds andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and/or vegetable oils. Proper fluidity may be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and/or by the use ofsurfactants. The prevention of the action of microorganisms can befacilitated by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580, which is hereinincorporated by reference in its entirety). Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. Moreover, for human administration, preparations willpreferably meet sterility, pyrogenicity, and the general safety andpurity standards as required by FDA Office of Biologics standards.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” relatesto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the adenovirus vectors of the invention may beadministered in conjunction with one or more immunostimulants, such asan adjuvant. An immunostimulant refers to essentially any substance thatenhances or potentiates an immune response (antibody and/orcell-mediated) to an antigen. One type of immunostimulant comprises anadjuvant. Many adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Certainadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF, IFN-γ, TNFα, IL-2, IL-8, IL-12,IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13, andothers, like growth factors, may also be used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient may support an immune response that includes Th1-and/or Th2-type responses. Within certain embodiments, in which aresponse is predominantly Th1-type, the level of Th1-type cytokines willincrease to a greater extent than the level of Th2-type cytokines. Thelevels of these cytokines may be readily assessed using standard assays.For a review of the families of cytokines, see Mosmann and Coffman, Ann.Rev. Immunol. 7:145-173, 1989. Thus, various embodiments of theinvention relate to therapies raising an immune response against atarget antigen, for example CEA, using cytokines, e.g. IFN-γ, TNFα IL-2,IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, and/orIL-13 supplied concurrently with a replication defective adenovirusvector treatment. In some embodiments, a cytokine or a nucleic acidencoding a cytokine, is administered together with a replicationdefective adenovirus described herein. In some embodiments, cytokineadministration is performed prior or subsequent to adenovirus vectoradministration. In some embodiments, a replication defective adenovirusvector capable of raising an immune response against a target antigen,for example CEA, further comprises a sequence encoding a cytokine.

Certain illustrative adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from GlaxoSmithKlein(Research Triangle Park, N.C.; see, for example, U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094, each incorporated hereinby reference in its entirety). CpG-containing oligonucleotides (in whichthe CpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462, each incorporated herein by reference in its entirety.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352, 1996. Another adjuvant for use in the presentinvention comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponinsOther formulations may include more than one saponin in the adjuvantcombinations of the present invention, for example combinations of atleast two of the following group comprising QS21, QS7, Quil A, β-escin,or digitonin.

In certain embodiments, the compositions may be delivered by intranasalsprays, inhalation, and/or other aerosol delivery vehicles. The deliveryof drugs using intranasal microparticle resins (Takenaga et al., JControlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,incorporated herein by reference in its entirety) are well-known in thepharmaceutical arts. Likewise, illustrative transmucosal drug deliveryin the form of a polytetrafluoroetheylene support matrix is described inU.S. Pat. No. 5,780,045, incorporated herein by reference in itsentirety.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit. Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol. Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). Liposomes have been used effectivelyto introduce genes, various drugs, radiotherapeutic agents, enzymes,viruses, transcription factors, allosteric effectors and the like, intoa variety of cultured cell lines and animals. Furthermore, the use ofliposomes does not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28, incorporatedherein by reference in its entirety). To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) may be designed using polymers able to be degraded invivo. Such particles can be made as described, for example, by Couvreuret al., Crit. Rev Ther Drug Carrier Syst. 1988; 5(1):1-20; zur Muhlen etal., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. JControlled Release. 1998 Jan. 2; 50(1-3):31-40; and U.S. Pat. No.5,145,684, each incorporated herein by reference in its entirety.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, may vary from individual toindividual, and from disease to disease, and may be readily establishedusing standard techniques. In general, the pharmaceutical compositionsand vaccines may be administered by injection (e.g., intracutaneous,intramuscular, intravenous or subcutaneous), intranasally (e.g., byaspiration), in pill form (e.g. swallowing, suppository for vaginal orrectal delivery). In certain embodiments, between 1 and 10 doses may beadministered over a 52 week period. In certain embodiments, 6 doses areadministered, at intervals of 1 month, and further booster vaccinationsmay be given periodically thereafter. Alternate protocols may beappropriate for individual patients. As such, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses may beadministered over a 1 year period or over shorter or longer periods,such as over 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100week periods. Doses may be administered at 1, 2, 3, 4, 5, or 6 weekintervals or longer intervals.

A suitable dose is an amount of an adenovirus vector that, whenadministered as described above, is capable of promoting a targetantigen immune response as described elsewhere herein. In certainembodiments, the immune response is at least 10-50% above the basal(i.e., untreated) level. In certain embodiments, the immune response isat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 125, 150, 200, 250, 300, 400,500 or more over the basal level. Such response can be monitored bymeasuring the target antigen(s) antibodies in a patient or byvaccine-dependent generation of cytolytic effector cells capable ofkilling patient tumor or infected cells in vitro, or other methods knownin the art for monitoring immune responses. Such vaccines should also becapable of causing an immune response that leads to an improved clinicaloutcome of the disease in question in vaccinated patients as compared tonon-vaccinated patients. In some embodiments, the improved clinicaloutcome comprises treating disease, reducing the symptoms of a disease,changing the progression of a disease, or extending life.

In general, an appropriate dosage and treatment regimen provides theadenovirus vectors in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome for the particular disease being treated intreated patients as compared to non-treated patients. The monitoringdata can be evaluated over time. In some embodiments, the progression ofa disease over time is altered. Such improvements in clinical outcomewould be readily recognized by a treating physician. Increases inpreexisting immune responses to a target protein can generally correlatewith an improved clinical outcome. Such immune responses may generallybe evaluated using standard proliferation, cytotoxicity or cytokineassays, which may be performed using samples obtained from a patientbefore and after treatment.

While one advantage of the present invention is the capability toadminister multiple vaccinations with the same or different adenovirusvectors, particularly in individuals with preexisting immunity to Ad,the adenoviral vaccines of this invention may also be administered aspart of a prime and boost regimen. A mixed modality priming and boosterinoculation scheme may result in an enhanced immune response. Thus, oneaspect of this invention is a method of priming a subject with a plasmidvaccine, such as a plasmid vector comprising a target antigen ofinterest, by administering the plasmid vaccine at least one time,allowing a predetermined length of time to pass, and then boosting byadministering the adenovirus vector. Multiple primings, e.g., 1-4, maybe employed, although more may be used. The length of time betweenpriming and boost may typically vary from about four months to a year,but other time frames may be used. In certain embodiments, subjects maybe primed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times with plasmidvaccines, and then boosted 4 months later with the adenovirus vector.

Patient Selection

Various embodiments of the invention relate to compositions and methodsfor raising an immune response against one or more CEA antigens inselected patient populations. Accordingly, methods and compositions ofthe invention may target patients with a cancer including but notlimited to carcinomas or sarcomas such as neurologic cancers, melanoma,non-Hodgkin's lymphoma, Hodgkin's disease, leukemias, plasmocytomas,adenomas, gliomas, thymomas, breast cancer, prostate cancer, colo-rectalcancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreaticcancer, esophageal cancer, lung cancer, ovarian cancer, cervical cancer,testicular cancer, gastric cancer, multiple myeloma, hepatoma, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), orother cancers can be targeted for therapy. In some cases, the targetedpatient population may be limited to individuals having colorectaladenocarcinoma, metastatic colorectal cancer, advanced CEA expressingcolorectal cancer breast cancer, lung cancer, bladder cancer, orpancreas cancer. A histologically confirmed diagnosis of a selectedcancer, for example colorectal adenocarcinoma, may be used. A particulardisease stage or progression may be selected, for example, patients withone or more of a metastatic, recurrent, stage III, or stage IV cancermay be selected for therapy with the methods and compositions of theinvention. In some embodiments, patients may be required to havereceived and, optionally, progressed through other therapies includingbut not limited to fluoropyrimidine, irinotecan, oxaliplatin,bevacizumab, cetuximab, or panitumumab containing therapies. In somecases, individual's refusal to accept such therapies may allow thepatient to be included in a therapy eligible pool with methods andcompositions of the invention. In some embodiments, individuals toreceive therapy using the methods and compositions of the invention maybe required to have an estimated life expectancy of at least, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 21, or 24 months. The patientpool to receive a therapy using the methods and compositions of theinvention may be limited by age. For example, individuals who are olderthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 25, 30, 35, 40, 50, 60, or more years old can be eligible fortherapy with methods and compositions of the invention. For anotherexample, individuals who are younger than 75, 70, 65, 60, 55, 50, 40,35, 30, 25, 20, or fewer years old can be eligible for therapy withmethods and compositions of the invention.

In some embodiments, patients receiving therapy using the methods andcompositions of the invention are limited to individuals with adequatehematologic function, for example with one or more of a WBC count of atleast 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more permicroliter, a hemoglobin level of at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or higher g/dL, a platelet count of at least 50,000; 60,000;70,000; 75,000; 90,000; 100,000; 110,000; 120,000; 130,000; 140,000;150,000 or more per microliter; with a PT-INR value of less than orequal to 0.8, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2.0, 2.5, 3.0, orhigher, a PTT value of less than or equal to 1.2, 1.4, 1.5, 1.6, 1.8,2.0×ULN or more. In various embodiments, hematologic function indicatorlimits are chosen differently for individuals in different gender andage groups, for example 0-5, 5-10, 10-15, 15-18, 18-21, 21-30, 30-40,40-50, 50-60, 60-70, 70-80 or older than 80.

In some embodiments, patients receiving therapy using the methods andcompositions of the invention are limited to individuals with adequaterenal and/or hepatic function, for example with one or more of a serumcreatinine level of less than or equal to 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 mg/dL or more, a bilirubinlevel of 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2 mg/dL or more, while allowing a higher limit for Gilbert'ssyndrome, for example, less than or equal tol.5, 1.6, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, or 2.4 mg/dL, an ALT and AST value of less than or equalto less than or equal to 1.5, 2.0, 2.5, 3.0×upper limit of normal (ULN)or more. In various embodiments, renal or hepatic function indicatorlimits are chosen differently for individuals in different gender andage groups, for example 0-5, 5-10, 10-15, 15-18, 18-21, 21-30, 30-40,40-50, 50-60, 60-70, 70-80 or older than 80.

In some embodiments, the K-ras mutation status of individuals who arecandidates for a therapy using the methods and compositions of theinvention can be determined. Individuals with a preselected K-rasmutational status can be included in an eligible patient pool fortherapies using the methods and compositions of the invention.

In various embodiments, patients receiving therapy using the methods andcompositions of the invention are limited to individuals withoutconcurrent cytotoxic chemotherapy or radiation therapy, a history of, orcurrent, brain metastases, a history of autoimmune disease, such as butnot restricted to, inflammatory bowel disease, systemic lupuserythematosus, ankylosing spondylitis, scleroderma, multiple sclerosis,thyroid disease and vitiligo, serious intercurrent chronic or acuteillness, such as cardiac disease (NYHA class III or IV), or hepaticdisease, a medical or psychological impediment to probable compliancewith the protocol, concurrent (or within the last 5 years) secondmalignancy other than non-melanoma skin cancer, cervical carcinoma insitu, controlled superficial bladder cancer, or other carcinoma in situthat has been treated, an active acute or chronic infection including: aurinary tract infection, HIV (e.g. as determined by ELISA and confirmedby Western Blot), and chronic hepatitis, or concurrent steroid therapy(or other immuno-suppressives, such as azathioprine or cyclosporin A).In some cases, patients with at least 3, 4, 5, 6, 7, 8, 9, or 10 weeksof discontinuation of any steroid therapy (except that used aspre-medication for chemotherapy or contrast-enhanced studies) may beincluded in a pool of eligible individuals for therapy using the methodsand compositions of the invention.

In some embodiments, patients receiving therapy using the methods andcompositions of the invention include individuals with thyroid diseaseand vitiligo.

Pre-Treatment Evaluation

In various embodiments, samples, for example serum or urine samples,from the individuals or candidate individuals for a therapy using themethods and compositions of the invention may be collected. Samples maybe collected before, during, and/or after the therapy for example,within 2, 4, 6, 8, 10 weeks prior to the start of the therapy, within 1week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeksfrom the start of the therapy, within 2, 4, 6, 8, 10 weeks prior to thestart of the therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks,6 weeks, 8 weeks, 9 weeks, or 12 weeks from the start of the therapy, in1 week, 10 day, 2 week, 3 week, 4 week, 6 week, 8 week, 9 week, or 12week intervals during the therapy, in 1 month, 3 month, 6 month, 1 year,2 year intervals after the therapy, within 1 month, 3 months, 6 months,1 year, 2 years, or longer after the therapy, for a duration of 6months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer. The samples maybe tested for any of the hematologic, renal, or hepatic functionindicators described herein as well as suitable others known in the art,for example a β-HCG for women with childbearing potential. In thatregard, hematologic and biochemical tests, including cell blood countswith differential, PT, INR and PTT, tests measuring Na, K, Cl, CO₂, BUN,creatinine, Ca, total protein, albumin, total bilirubin, alkalinephosphatase, AST, ALT and glucose are within the bounds of theinvention. In some embodiments, the presence or the amount of HIVantibody, Hepatitis BsAg, or Hepatitis C antibody are determined in asample from individuals or candidate individuals for a therapy using themethods and compositions of the invention. Biological markers, such asantibodies to CEA or the neutralizing antibodies to Ad5 vector can betested in a sample, such as serum, from individuals or candidateindividuals for a therapy using the methods and compositions of theinvention. In some cases, one or more samples, such as a blood samplecan be collected and archived from an individuals or candidateindividuals for a therapy using the methods and compositions of theinvention. Collected samples can be assayed for immunologic evaluation.Individuals or candidate individuals for a therapy using the methods andcompositions of the invention can be evaluated in imaging studies, forexample using CT scans or MRI of the chest, abdomen, or pelvis. Imagingstudies can be performed before, during, or after therapy using themethods and compositions of the invention, during, and/or after thetherapy, for example, within 2, 4, 6, 8, 10 weeks prior to the start ofthe therapy, within 1 week, 10 day, 2 weeks, 3 weeks, 4 weeks, 6 weeks,8 weeks, or 12 weeks from the start of the therapy, within 2, 4, 6, 8,10 weeks prior to the start of the therapy, within 1 week, 10 day, 2weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 9 weeks, or 12 weeks from thestart of the therapy, in 1 week, 10 day, 2 week, 3 week, 4 week, 6 week,8 week, 9 week, or 12 week intervals during the therapy, in 1 month, 3month, 6 month, 1 year, 2 year intervals after the therapy, within 1month, 3 months, 6 months, 1 year, 2 years, or longer after the therapy,for a duration of 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years orlonger.

Treatment Plans

Dosage and Administration

Compositions and methods of the invention contemplate various dosage andadministration regimens during therapy. Patients may receive areplication defective adenovirus or adenovirus vector, for example Ad5[E1-, E2B-]-CEA(6D), that is capable of raising an immune response in anindividual against a target antigen described herein, for example CEA.In various embodiments, the replication defective adenovirus isadministered at a dose that suitable for effecting such immune response.In some cases, the replication defective adenovirus is administered at adose that is greater than or equal to 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.5×10¹², 2×10¹², 3×10¹², ormore virus particles (VP) per immunization. In some cases, thereplication defective adenovirus is administered at a dose that is lessthan or equal to 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, 1×10¹², 1.5×10¹², 2×10¹², 3×10¹², or more virus particles perimmunization. In various embodiments, a desired dose described herein isadministered in a suitable volume of formulation buffer, for example avolume of about 0.1-10 ml, 0.2-8 ml, 0.3-7 ml, 0.4-6 ml, 0.5-5 ml, 0.6-4ml, 0.7-3 ml, 0.8-2 ml, 0.9-1.5 ml, 0.95-1.2 ml, 1.0-1.1 ml. Those ofskill in the art appreciate that the volume may fall within any rangebounded by any of these values (e.g., about 0.5 ml to about 1.1 ml. Theadministration of the virus particles can be through a variety ofsuitable paths for delivery, for example it can be by injection (e.g.,intracutaneous, intramuscular, intravenous or subcutaneous),intranasally (e.g., by aspiration), in pill form (e.g. swallowing,suppository for vaginal or rectal delivery. In some embodiments, asubcutaneous delivery may be preferred and can offer greater access todendritic cells.

The administration of the virus particles to an individual may berepeated. The repeated deliveries of virus particles may follow aschedule or alternatively, may be performed on an as needed basis. Forexample, the individual's immunity against a target antigen, for exampleCEA, may be tested and replenished as necessary with additionaldeliveries. In some embodiments, schedules for delivery includeadministrations of virus particles at regular intervals. Joint deliveryregimens may be designed comprising one or more of a period with aschedule and/or a period of need based administration assessed prior toadministration. For example, a therapy regimen may include anadministration, for example subcutaneous administration once every threeweeks then another immunotherapy treatment every three months untilremoved from therapy for any reason including death. Another exampleregimen comprises three administrations every three weeks then anotherset of three immunotherapy treatments every three months. Anotherexample regimen comprises a first period with a first number ofadministrations at a first frequency, a second period with a secondnumber of administrations at a second frequency, a third period with athird number of administrations at a third frequency etc. and optionallyone or more periods with undetermined number of administrations on an asneeded basis. The number of administrations in each period can beindependently selected and can for example be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. The frequency of theadministration in each period can also be independently selected, canfor example be about every day, every other day, every third day, twicea week, once a week, once every other week, every three weeks, everymonth, every six weeks, every other month, every third month, everyfourth month, every fifth month, every sixth month, once a year etc. Thetherapy can take a total period of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36 months ormore. The scheduled interval between immunizations may be modified sothat the interval between immunizations is revised by up to a fifth, afourth, a third, or half of the interval. For example, for a 3-weekinterval schedule, an immunization may be repeated between 20 and 28days (3 weeks −1 day to 3 weeks +7 days). For the first 3 immunizations,if the second and/or third immunization is delayed, the subsequentimmunizations may be shifted allowing a minimum amount of buffer betweenimmunizations. For example, for a three week interval schedule, if animmunization is delayed, the subsequent immunization may be scheduled tooccur no earlier than 17, 18, 19, or 20 days after the previousimmunization.

Compositions of the invention, such as Ad5 [E1-, E2b-]-CEA(6D) virusparticles, can be provided in various states, for example, at roomtemperature, on ice, or frozen. Compositions may be provided in acontainer of a suitable size, for example a vial of 2 ml vial. In oneembodiment, 1 2 ml vial with 1.0 ml of extractable vaccine contains5×10¹¹ total virus particles/mL. Storage conditions includingtemperature and humidity may vary. For example, compositions for use intherapy may be stored at room temperature, 4° C., −20° C. or lower untilused.

General Evaluations

In various embodiments, general evaluations are performed on theindividuals receiving treatment according to the methods andcompositions of the invention. One or more of any tests may be performedas needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. Adifferent set of tests may be performed concurrent with immunization vs.at time points without immunization.

General evaluations may include one or more of medical history, ECOGPerformance Score, Karnofsky performance status, and complete physicalexamination with weight by the attending physician. Any othertreatments, medications, biologics, or blood products that the patientis receiving or has received since the last visit may be recorded.Patients may be followed at the clinic for a suitable period, forexample approximately 30 minutes, following receipt of vaccine tomonitor for any adverse reactions. Local and systemic reactogenicityafter each dose of vaccine will may be assessed daily for a selectedtime, for example for 3 days (on the day of immunization and 2 daysthereafter). Diary cards may be used to report symptoms and a ruler maybe used to measure local reactogenicity. Immunization injection sitesmay be assessed. CT scans or MRI of the chest, abdomen, and pelvis maybe performed.

Hematological and Biochemical Assessment

In various embodiments, hematological and biochemical evaluations areperformed on the individuals receiving treatment according to themethods and compositions of the invention. One or more of any tests maybe performed as needed or in a scheduled basis, such as on weeks 0, 3, 6etc. A different set of tests may be performed concurrent withimmunization vs. at time points without immunization.

Hematological and biochemical evaluations may include one or more ofblood test for chemistry and hematology, CBC with differential, Na, K,Cl, CO₂, BUN, creatinine, Ca, total protein, albumin, total bilirubin,alkaline phosphatase, AST, ALT, glucose, and ANA.

Biological Markers

In various embodiments, biological markers are evaluated on individualsreceiving treatment according to the methods and compositions of theinvention. One or more of any tests may be performed as needed or in ascheduled basis, such as on weeks 0, 3, 6 etc. A different set of testsmay be performed concurrent with immunization vs. at time points withoutimmunization.

Biological marker evaluations may include one or more of measuringantibodies to CEA or the Ad5 vector, from a serum sample of adequatevolume, for example about 5 ml Biomarkers (e.g., CEA or CA15-3) may bereviewed if determined and available

Immunological Assessment

In various embodiments, an immunological assessment is performed onindividuals receiving treatment according to the methods andcompositions of the invention. One or more of any tests may be performedas needed or in a scheduled basis, such as on weeks 0, 3, 6 etc. Adifferent set of tests may be performed concurrent with immunization vs.at time points without immunization.

Peripheral blood, for example about 90 mL may be drawn prior to eachimmunization and at a time after at least some of the immunizations, todetermine whether there is an effect on the immune response at specifictime points during the study and/or after a specific number ofimmunizations. Immunological assessment may include one or more ofassaying peripheral blood mononuclear cells (PBMC) for T cell responsesto CEA using ELISpot, proliferation assays, multi-parameter flowcytometric analysis, and cytoxicity assays. Serum from each blood drawmay be archived and sent and determined.

Tumor Assessment

In various embodiments, a tumor assessment is performed on individualsreceiving treatment according to the methods and compositions of theinvention. One or more of any tests may be performed as needed or in ascheduled basis, such as prior to treatment, on weeks 0, 3, 6 etc. Adifferent set of tests may be performed concurrent with immunization vs.at time points without immunization.

Tumor assessment may include one or more of CT or MRI scans of chest,abdomen, or pelvis performed prior to treatment, at a time after atleast some of the immunizations and at approximately every three monthsfollowing the completion of a selected number, for example 2, 3, or 4,of first treatments and for example until removal from treatment.

Rate of Immune Response

Immune responses against a target antigen described herein, such as CEA,may be evaluated from a sample, such as a peripheral blood sample of anindividual using one or more suitable tests for immune response, such asELISpot, cytokine flow cytometry, or antibody response. A positiveimmune response by ELISpot is described at the 2002 Society of BiologicTherapy Workshop on “Immunologic Monitoring of Cancer Vaccine Therapy”,i.e. a T cell response is considered positive if the mean number ofspots adjusted for background in six wells with antigen exceeds thenumber of spots in six control wells by 10 and the difference betweensingle values of the six wells containing antigen and the six controlwells is statistically significant at a level of p<0.05 using theStudent's t test. Immunogenicity assays may occur prior to eachimmunization and at scheduled timepoints during the period of thetreatment. For example, a timepoint for an immunugenecity assay ataround week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20,24, 30, 36, or 48 of a treatment may be scheduled even without ascheduled immunization at this time. In some cases, an individual may beconsidered evaluable for immune response if they receive at least aminimum number of immunizations, for example 1, 2, 3, 4, 5, 6, 7, 8, 9,or more immunizations.

Determination of Clinical Response

In some embodiments, disease progression or clinical responsedetermination is made according to the RECIST 1.1 criteria amongpatients with measurable/evaluable disease.

In some embodiments, therapies using the methods and compositions of theinvention affect a Complete Response (CR; disappearance of all targetlesions for target lesions or disappearance of all non-target lesionsand normalization of tumor marker level for non-target lesions) in anindividual receiving the therapy.

In some embodiments, therapies using the methods and compositions of theinvention affect a Partial Response (PR; at least a 30% decrease in thesum of the LD of target lesions, taking as reference the baseline sum LDfor target lesions) in an individual receiving the therapy.

In some embodiments, therapies using the methods and compositions of theinvention affect a Stable Disease (SD; neither sufficient shrinkage toqualify for PR nor sufficient increase to qualify for PD, taking asreference the smallest sum LD since the treatment started for targetlesions) in an individual receiving the therapy.

In some embodiments, therapies using the methods and compositions of theinvention affect an Incomplete Response/Stable Disease (SD; persistenceof one or more non-target lesion(s) or/and maintenance of tumor markerlevel above the normal limits for non-target lesions) in an individualreceiving the therapy.

In some embodiments, therapies using the methods and compositions of theinvention affect a Progressive Disease (PD; at least a 20% increase inthe sum of the LD of target lesions, taking as reference the smallestsum LD recorded since the treatment started or the appearance of one ormore new lesions for target lesions or persistence of one or morenon-target lesion(s) or/and maintenance of tumor marker level above thenormal limits for non-target lesions) in an individual receiving thetherapy.

EXAMPLES Example 1 Multiple Injections of Ad5Null Adenovirus VectorProduces Anti-Adenovirus Antibodies

This example shows that multiple injections of Ad5-null results in theproduction of anti-adenovirus antibodies in the injected subjects

It was demonstrated that the Ad5Null adenovirus vector that does notcontain any heterologous nucleic acid sequences, generates aneutralizing immune response in mice. In particular, in one experiment,female Balb/c mice aged 5-7 weeks were immunized with Ad5Null viralparticles at 14 day intervals. To determine the presence ofanti-adenovirus antibodies, an enzyme linked immunosorbent assay (ELISA)was used. For this ELISA, 10⁹ viral particles were coated ontomicrotiter wells in 100 μL of 0.05M carbonate/bicarbonate buffer, pH9.6, and incubated overnight at room temperature. For a standardimmunoglobulin G (IgG) reference curve, 200 ng, 100 ng, 50 ng, 25 ng,and 0 ng of purified mouse IgG (Sigma Chemicals) were coated ontomicrotiter wells as described above. After incubation, all wells werewashed 3 times with 250 μl of 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS), pH 7.4. After washing, 250 μl of BSA/PBS wasadded to all and incubated for 30 minutes at room temperature to blockunbound sites. After incubation, all wells were washed 3 times with 250μL of BSA/PBS. After washing, 200 μl of a 1/100 serum dilution inBSA/PBS was added to wells and incubated for 1 hour at room temperature.For a positive control, 200 μl of a 1/10000 dilution of anti-adenovirusantiserum (Biodesign International) in BSA/PBS were added to wells.Control wells contained BSA/PBS only. After incubation, all wells werewashed 3 times with 250 μl of BSA/PBS. After washing, 200 μL of a1/10000 dilution of peroxidase conjugated gamma chain specific goatanti-mouse IgG (Sigma Chemicals) in BSA/PBS were added to each well andincubated for 1 hour at room temperature. After incubation, all wellswere washed 3 times with 250 μL of BSA/PBS. After washing, 200.mu.L ofdeveloping reagent (0.5 mg/mL 1,2 phenylenediamine in 0.2M potassiumphosphate buffer, pH 5.0, containing 0.06% hydrogen peroxide) was addedto each well and incubated for 30-40 minutes at room temperature. Afterincubation, the color reaction was stopped by addition of 50.mu.L 5N HClto each well. All wells were then read in a microwell plate reader at492 nm. After readings were obtained, the optical density readings ofunknown samples were correlated with the standard IgG curve to obtainthe nanograms of IgG bound per well. This was performed using the INSTATstatistical package.

As shown in FIG. 1, significant levels (P<0.001) of anti-adenovirus IgGantibody were detected in mice 2 weeks after a first injection with 10¹⁰Ad-5-null. A significantly higher level (P<0.001) was observed 2 weeksafter a second injection with 10¹⁰ adenovirus. Significantly higher(P<0.001) levels of antibody were continued to be observed 2 weeks aftera third injection with 10¹⁰ Ad5-null. Each value represents the averageof triplicate determinations from pooled sera of 5 mice in each group.These results indicate that multiple injections of Ad5-null results inthe production of anti-adenovirus antibodies in the injected subjects.

To determine the presence of neutralizing antibody to Ad, the followingassay was utilized. A HEK-293T cell line was cultured in 200.mu.L ofculture medium consisting of DMEM containing 10% fetal calf serum(DMEM/FCS) in microwell tissue culture plates at a cell concentration of2.times.10³ cells per well for 24 hours at 37 C in 5% CO.sub.2. Afterincubation, 100.mu.L of culture medium was removed from triplicate wellsand mixed with 20.mu.L of DMEM/FCS containing viral particles (VP).After mixing, the 120.mu.L mixture was added back to the respectivemicrowells. In another set of triplicate wells, 100.mu.L of culturemedium was removed and mixed with 20.mu.L of heat inactivated (56 C for1 hour) Ad immune mouse serum previously incubated with VP for one hourat room temperature. After mixing, the 120.mu.L mixture was added backto the respective wells. In triplicate cell control wells, 20.mu.L ofDMEM/FCS was added to control for total culture medium volume.Triplicate medium only control wells contained 220.mu.L of DMEM/FCS. Thetissue culture plate was incubated for an additional 3 days at 37 C in5% CO.sub.2. After incubation, 40.mu.L of PROMEGA cell viability reagent(Owen's reagent) was added to all wells and incubated for 75 minutes at37 C in 5% CO.sub.2. In this assay, the Owen's reagent (MTS tetrazoliumcompound) is bioreduced by viable cells into a colored formazan productthat is soluble in tissue culture medium. The quantity of formazanproduct as measured by absorbance at 490 nm is directly proportional tothe number of living cells in culture. After incubation, 150.mu.L wasremoved from each well and transferred to another microwell plate foroptical density readings. Optical density readings at 492 nm weresubsequently obtained using a microwell plate reader.

In an experiment to detect the presence of neutralizing antibodies toAd, groups of 5 mice each were injected once, twice, or three times with10¹⁰ Ad5null at two week intervals. Two weeks after the final injectionof virus, mice were bled, pooled, and assessed for neutralizing antibodyas described above using 4.times.10⁷ VP incubated with or without heatinactivated sera. Cells cultured alone served as a control group. Asshown in FIG. 2, normal mice and mice injected one time with AdSnull didnot exhibit significant levels of neutralizing antibody. Mice injectedtwo times with Ad exhibited significant (P<0.05) levels of neutralizingantibody as compared with cells incubated with virus only. Mice injectedthree times with Ad5-null also exhibited significant (P<0.01) levels ofneutralizing antibody as compared with cells incubated with virus only.

Example 2 The Ad5 [E1-]-CEA Vector Vaccine Induces CEA Specific ImmuneResponse Upon Re-Immunization in Ad5 Immune Mice

This example shows that the Ad5 [E1-, E2b-] vector platform induces CMIresponses against the tumor associated antigen (TAA) carcinoembryonicantigen (CEA) in the presence of pre-existing Ad5 immunity in mice.

Characterization of Ad5 CEA Vectors

Initial studies were performed to confirm CEA gene expression of twoAd5-CEA vector platforms. It was first determined that the CEA antigencould be expressed on cells transfected with the vaccine vectorplatforms. A549 cells were obtained from ATCC and transfected with Ad5[E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Western blot analysis revealed thatcells transfected with the vector platforms expressed CEA antigen.

Induction of Ad5 Immunity in Mice

To assess the levels of Ad5 immunity that could be induced, groups ofAd5 naive C57BI/6 mice were injected subcutaneously with the Ad5 vectorplatform (VP). Twenty eight to forty two days later, serum samples werecollected and assessed for endpoint Ad5 NAb titers. As shown in FIG. 3,undetectable Ad5 NAb titers (endpoint Ad5 NAb titer <1/25) were observedin normal control mice. Ad5 NAb (endpoint titers of 1/25 to 1/50) wasdetectable after one injection but dramatically increased after threeinjections of 10¹⁰ Ad5. Therefore, in additional Ad5 immune studies,mice were injected twice with 10¹⁰ Ad5 VP to render the animals Ad5immune.

Immunization of Ad5 Immune Mice with Ad5 [EH-CEA or Ad5 [E1-, E2b-]-CEA.

These experiments were designed to determine and compare theimmunization induction potential of Ad5 [E1-]-CEA and Ad5 [E1-,E2b-]-CEA vaccines in Ad5 immune mice. Groups of female C57BI/6 mice, 4to 8 weeks old, were immunized 2 times at 2 week intervals with 10¹⁰Ad5-null VP. Two weeks following the last Ad5-null immunization, themice were immunized 3 times at weekly intervals with 10¹⁰ VP of Ad5[E1-]-CEA or Ad5 [E1-, E2b-]-CEA. Two weeks following the lastimmunization, mice were euthanized and their spleens and sera harvestedfor analyses.

CMI responses were assessed by ELISpot assays performed on splenocytesexposed to intact CEA antigen. Splenocytes from Ad5 immune C57BI/6 micethat were immunized subcutaneously with Ad5 E1-]-CEA or Ad5 [E1-,E2b-]-CEA were harvested and assessed for the number of IFN-γ and IL-2secreting cells as described above. As shown in FIGS. 4A and 4B,significantly elevated numbers of both IFN-γ and IL-2 secreting cellswere observed in spleens assayed from mice immunized with Ad5 [E1-,E2b-]-CEA as compared to immunized Ad5 [E1-]-CEA mice. Specificitystudies revealed that immunizations with Ad5 CEA vectors inducedspecific CEA associated CMI responses and not responses against otherirrelevant antigens such as the HIV-gag protein or β-galactosidase.These results demonstrate that immunization of Ad5 immune mice with Ad5[E1-, E2b-]-CEA induce significantly higher CMI responses.

Lack of Adverse Liver Effects in Immunized Mice

Toxicity studies were performed on serum from Ad5 immune female C57BI/6mice immunized with Ad5 [E1-]-CEA, Ad5 [E1-, E2b-]-CEA as describedabove. Ad5 naive or Ad5 immune mice injected with buffer alone served ascontrols. Three days after the third immunization, aspartateaminotransferase (AST) levels were assessed on the blood samples todetermine liver toxicity due to the treatment. AST levels were notelevated over controls following immunization with either vector (FIG.5). Alanine aminotransferase (ALT) levels were also assessed and similarresults were observed.

Ad5 [E1-, E2b-]-CEA Immunotherapy in Ad5 Immune Tumor Bearing Mice

Based upon the successful immunological results observed above, studiesin which MC38 tumors were established in mice and then treated wereperformed as described below. For these studies a CEA expressing MC38murine cell line was used. This cell line has been genetically modifiedto express human CEA and can be implanted into C57BI/6 mice. After tumorestablishment, the mice were treated with the novel Ad5 [E1-, E2b-]-CEAvector platform. To determine if Ad5 immune tumor bearing mice could betreated with the Ad5 [E1-, E2b-]-CEA vector, C57BI/6 mice were injectedtwo times subcutaneously with 10¹⁰ Ad5 [E1-]-null VP at 14 day intervalsto render the mice Ad5 immune. Two weeks after the last injection, twogroups of 7 C57BI/6 mice were injected subcutaneously with 10⁶ CEAexpressing MC38 tumor cells. Seven days later, when tumors werepalpable, one group of mice was treated by distal subcutaneous injectionwith 10¹⁰ VP of Ad5 [E1-, E2b-]-CEA on days 7, 13 and 19. A group of 7injection buffer only treated C57BI/6 mice served as untreated controls.All mice were monitored for tumor size over a 21 day period and tumorvolumes were determined as previously described.

As shown in FIG. 6, the tumor growth by day 19 was significantly reducedin the Ad5 [E1-, E2b-]-CEA treated mice and remained so. At the end ofthe study (Day 22), the mice were sacrificed and the tumors were excisedand weighed. As shown in FIG. 7, the tumors in the mice treated with Ad5[E1-, E2b-]-CEA were significantly (P<0.05) smaller in weight than theuntreated controls.

At the termination of the study, spleens were collected from mice andthe CEA specific CMI response was determined by ELISpot assay. CEAspecific IFN-γ secretion response was significantly higher in miceimmunized with Ad5 [E1-, E2b-]-CEA than in mice who received MC-38 tumorcells alone. These results indicate that treatment of CEA expressingtumors in Ad5 immunized mice using the Ad5 [E1-, E2b-]-CEA vaccine cansignificantly decrease tumor growth progression.

Example 3 Quantitative ELISA for CEA Expression on A549 Cells afterInfection Experimental Design

On day one, of the assay a BD Falcon Tissue Culture 96-well plate wasseeded with A549 cells passaged three days prior (lot#30Jul02, passagep+23), (7.7×10³ cells/well) and placed into a 37±2° C. incubator with a5±2% CO₂ atmosphere overnight. The next day, a dilution series of thetest article were prepared and replicate wells were inoculated at levelsranging from 1.56×10³ to 2.5×10⁴ viral particles/well. Untreated A549cells were used to serve as the mock sample. On day four of the assaywells were treated with a 10% Triton X-100 solution for analysis byELISA to measure CEA concentration.

For the ELISA, a microtiter plate was coated overnight with an anti-CEAcapture antibody (abcam Carcino embryonic antigen CEA antibody[(NCRC16(AKA161)] catalog number ab2077, lot #201993. The wells werewashed to remove unbound reactants, and the plate was blocked with aPhosphate Buffered Saline (PBS) solution containing 1% Tween 20. to fallwithin the range of the standard curve. After the blocking period, thewells were washed, and samples, controls, and standards were incubatedin assigned triplicate wells. Unbound reactants were removed by washing,and a rabbit polyclonal to CEA detection antibody (abcam carcinoembryonic antigen CEA antibody catalog# ab15987, lot#898335) was added.After incubation, the wells were washed and incubated with3,31′,5,5′-tetramethylbenzidine (TMB), the peroxidase substrate. Thesubstrate formed a colored product in the presence of the enzyme,reaction was stopped with 1M phosphoric acid solution, and theabsorbance was determined on a calibrated microplate reader. Acalibration curve was generated from standards containing knownconcentrations of CEA, and the curve was used to determine theconcentration of CEA in the samples

Test Evaluation

The quantity of CEA produced per virus particle was calculated from theconcentration of CEA measured by ELISA, after adjusting for dilution andmultiplicity of infection (MOI). The value determined in a similarmanner for culture media alone was subtracted to compensate forbackground levels present in the media. The sample analysis is shown inTable 1.

TABLE 1 Sample Analysis A₄₅₀- ELISA AVG Total A₅₄₀ Blank Dil'n CEA CEACEA CEA Sample ID Rep 1 Rep 2 Mean SD RSD Subtr Fact ng/ml ng/ml ng/mlNg/vp 10-002917 Well 1 at 1.3387 1.2977 1.318 0.029 2.2% 1.244 10007,731 7,691 7,621 0.30 2.5E+04 vp 0.7121 0.6789 0.693 0.023 3.4% 0.6222000 7,797 10-002917 Well 2 at 1.1717 1.1329 1.152 0.027 2.4% 1.078 10006,563 2.5E+04 vp 0.6222 0.6151 0.619 0.005 0.8% 0.545 2000 6,97510-002917 Well 3 at 1.1659 2.0492 1.608 0.625 38.9% 1.534 1000 10,2642.5E+04 vp 0.6131 0.5946 0.604 0.013 2.2% 0.530 2000 6,815 10-002917Well 1 at 1.1051 1.0759 1.091 0.021 1.9% 1.017 1000 6,169 6,049 5,9790.48 1.25E+04 vp 0.5970 0.5716 0.584 0.018 3.1% 0.510 2000 6,60210-002917 Well 2 at 1.0652 1.0376 1.051 0.020 1.9% 0.977 1000 5,9191.25E+04 vp 0.5726 0.5770 0.575 0.003 0.5% 0.501 2000 6,506 10-002917Well 3 at 0.9731 0.9514 0.962 0.015 1.6% 0.888 1000 5,383 1.25E+04 vp0.5049 0.4970 0.501 0.006 1.1% 0.427 2000 5,716 10-002917 Well 1 at0.7601 0.7210 0.741 0.028 3.7% 0.667 1000 4,141 4,286 4,216 0.676.25E+03 vp 0.4041 0.3881 0.396 0.011 2.9% 0.322 2000 4,566 10-002917Well 2 at 0.7157 0.7068 0.711 0.006 0.9% 0.637 1000 3,979 6.25E+03 vp0.3893 0.3843 0.387 0.004 0.9% 0.313 2000 4,465 10-002917 Well 3 at0.7360 0.7188 0.727 0.012 1.7% 0.653 1000 4,065 6.25E+03 vp 0.39950.3807 0.390 0.013 3.4% 0.316 2000 4,499 10-002917 Well 1 at 0.89200.8878 0.890 0.003 0.3% 0.816 500 2,483 2,690 2,620 0.84 3.13E+03 vp0.4573 0.4613 0.459 0.003 0.6% 0.385 1000 2,631 10-002917 Well 2 at0.8615 0.8544 0.858 0.005 0.6% 0.784 500 2,393 3.13E+03 vp 0.4425 0.44060.442 0.001 0.3% 0.368 1000 2,538 10-002917 Well 3 at 1.0518 1.04641.049 0.004 0.4% 0.975 500 2,953 3.13E+03 vp 0.5519 0.5565 0.554 0.0030.6% 0.480 1000 3,141 10-002917 Well 1 at 1.8771 1.8616 1.869 0.011 0.6%1.795 100 1,351 1,271 1,201 0.77 1.56E+03 vp 1.1963 1.1695 1.183 0.0191.6% 1.109 200 1,354 10-002917 Well 2 at 1.7435 1.7436 1.744 0.000 0.0%1.670 100 1,179 1.56E+03 vp 1.0960 1.0788 1.087 0.012 1.1% 1.013 2001,229 10-002917 Well 3 at 1.7801 1.8098 1.795 0.021 1.2% 1.721 100 1,2451.56E+03 vp 1.1041 1.1263 1.115 0.016 1.4% 1.041 200 1,264 10-002917Well 1 1.2509 1.2278 1.239 0.016 1.3% 1.165 10 72 70 0 — Mock 0.71460.6952 0.705 0.014 1.9% 0.631 20 79 10-002917 Well 2 1.2290 1.2382 1.2340.007 0.5% 1.160 10 71 Mock 0.7246 0.7133 0.719 0.008 1.1% 0.645 20 8010-002917 Well 3 0.9769 0.9750 0.976 0.001 0.1% 0.902 10 55 Mock 0.55790.5454 0.552 0.009 1.6% 0.478 20 63

Example 4 Schedule, Dose, Route of Immunization Safety Data

Initial studies were performed to evaluate and confirm that an Ad5 [E1-,E2b-] vector platform could express the antigen proteins on transfectedcells. A-549 cells were transfected with vaccine platforms and analyzedby Western Blot Analysis. Antigen proteins such as HIV-gag, HIV-pol, orHIV-nef were observed to be expressed on cells once they weretransfected with the Ad5 [E1-, E2b-] vector platforms. A representativeWestern Blot is presented in FIG. 8.

A dose response evaluation was performed using the Ad5 [E1-, E2b-]vector platform and demonstrated that 10¹⁰ virus particles (VP) is adose that results in a desired CMI response against a transgene productin a murine model. CMI responses were assessed by utilizing an ELISpotassay to detect interferon-gamma (IFN-γ) and IL-2 secreting cells(splenocytes) from spleens of mice. Furthermore, in murine and non-humanprimate (NHP) models, three immunizations using 10¹⁰ VP separated by twoweeks to four weeks, respectively, resulted in the desired CMIresponses. In mice, a greater degree of CMI responses were observedafter multiple immunizations as compared with one immunization only(FIG. 9).

In a NHP model, the animals were rendered Ad5 immune by injection withwild type Ad5 virus. After detection of the presence of Ad5 neutralizingantibody, which confirmed that the animals were immune to Ad5, theanimals were vaccinated with an Ad5 [E1-, E2b-] vector platform threetimes at monthly intervals. As shown in FIG. 10, after immunizations,the presence of robust CMI responses was detected, when peripheral bloodmononuclear cells (PBMCs) of animals were assessed for IFN-γ and IL-2secreting cells.

In addition to the preliminary immunology studies performed in theinitial vaccine trial in 3 NHP shown above, toxicity studies were alsoperformed on the same NHP vaccinated with Ad5 [E1-, E2b-]-HIV gag.Animal temperatures and weights were assessed during the study period.The animals gained weight as they grew during the study period. Notemperature differences were observed during the study period.Hematology studies were also performed on the vaccinated NHP.

There appeared to be a small increase in the white blood cell count 2weeks after the second vaccination that normalized thereafter.

Other than fluctuation in values, there appeared to be no otherdifferences in hematology values during the course of the study.Chemistry values were also determined in the NHP during the course ofthe study. Alkaline phosphatase levels declined slightly during thecourse of the study but remained in the normal range. Albumin levelsdeclined slightly during the course of the study but remained in thenormal. There were no other differences observed in the bloodchemistries during the course of the study. The route of immunization inthis clinical study is chosen since the preponderance of DC reside inthe dermis.

A desired level of CMI response was induced using the Ad5 [E1-, E2b-]platform employing CEA and other transgenes. Using an Ad5 [E1-,E2b-]-CEA vector platform, both non-Ad5 immune and Ad5 pre-immunizedmice were injected three times with the vaccine. After immunizations,the splenocytes from mice were assessed by ELISpot for IFN-γ secretingcells. As shown in FIG. 11, elevated CMI responses were observed afterimmunizations and the levels of CMI responses were similar in bothnon-Ad5 immune and Ad5 pre-immunized mice. These results indicate thatrobust CMI responses can be induced despite the presence of pre-existingAd5 immunity. A III clinical study was designed using threeimmunizations separated by three weeks via a needle subcutaneousdelivery method.

Rationale for Schedule, Dose, Route of Administration

A clinical study design flowed from pre-clinical and clinical studies inanimals and humans using the Ad5 [E1-, E2b-] vector platform. A doseresponse evaluation using the Ad5 [E1-, E2b-] vector platform wasperformed demonstrating that 10¹⁰ virus particles (VP) is a dose whichresults in a desired CMI response against a transgene product in amurine model. Furthermore, in murine and non-human primate (NHP) modelsthree immunizations using 10¹⁰ VP separated by two to four weeksrespectively resulted in the desired CMI. The route of immunization ischosen since a preponderance of dendritic cells (DC) reside in thedermis. Using this premise, multiple murine and NHP studies wereperformed using a sub-cutaneous injection protocol. A desired level ofcirculating CMI was induced using the Ad5 [E1-, E2b-] platform employingCEA and other transgenes. A phase III clinical study followed usingthree immunizations separated by three weeks via a needle subcutaneous(SQ) delivery method continuing immunotherapy treatment every threemonths until removed from study for any reason including death.

Construction and Production of Ad5 [E1-, E2b-]-CEA(6D).

The cDNA sequence containing the modified CEA with the CAP1(6D) mutationwas produced. Clinical grade Ad5 [E1-, E2b-]-CEA(6D) was constructed aspreviously described (Gabitzsch et al. (2010) Anti-tumor immunitydespite immunity to adenovirus using a novel adenoviral vector Ad5 [E1-,E2b-]-CEA. Cancer Immunol Immunoother 59:1131-1135) and manufacturedusing the E.C7 cell line.

Summary

A total of 34 patients (32 colorectal cancer patients, one bladdercancer patient, and one lung cancer patient) were entered into the PhaseI/II clinical study under IND14325. The majority received all threescheduled immunotherapy treatments with ETBX-011(Ad5 [E1-,E2b-]-CEA(6D)). Five patients who stopped immunotherapy early did so dueto significant disease progression. RECIST 1.0 criteria using CT or MRIscans obtained at baseline and after treatments were completed. Toxicitywas assessed according to the National Cancer Institute CommonTerminology Criteria for Adverse Events (CTCAE) version 4.0. Peripheralblood carcinoembryonic antigen (CEA) levels, hematology, serumchemistries, and anti-nuclear antibody titers were compared betweenbaseline and 9 weeks following the initiation of immunotherapy. Survivalwas measured from the day of the first immunization until death from anycause.

A total of 94 treatments were administered to patients. No dose limitingtoxicity or serious adverse events (SAE) that resulted in treatmentdiscontinuation at any treatment dose level. There was only onesignificant change in a blood hematology value. As a group, the basophilcount was significantly lower at week 9, three weeks after treatmentended. However, this value remained in the normal range for basophilcounts and, overall, there appeared to be no significant biologicaleffect. With a median follow-up of 7.4 months, all 34 patients as agroup (cohorts 1, 2, 3/phase II, and cohort 5) experienced a 12-monthsurvival proportion of 41.4%. Of the 34 patients entered into the study,28 patients received the three immunotherapy treatments and experienceda 12-month survival proportion of 55%. For the colorectal adenocarcinomapatients, 27 patients received the three immunotherapy treatments andexperienced a 12-month survival proportion of 53%. A dose response toincreasing levels of ETBX-011 was observed with the highestcell-mediated immune (CMI) responses occurring in patients that receivedthe highest dose of 5×10¹¹ VP of ETBX-011. When the highest CEA specificCMI responses were compared with pre-existing or vector induced Ad5 NAbactivity, there was no correlation between levels of CEA specific CMIand Ad5 neutralizing antibody (NAb) level. These clinical trial datalead us to believe that there is sufficient data to advance to arandomized Phase III trial for the treatment of metastatic colorectaladenocarcinoma with overall survival as the clinical endpoint.

Protocol Schema and Patient Treatment.

The clinical study was performed under an FDA-approved InvestigationalNew Drug Exemption and registered at ClinicalTrials.gov. Participantswere recruited from medical oncology clinics at Duke University MedicalCenter, Durham, N.C. and Medical Oncology Associates, Spokane, Wash.Patients provided informed consent approved by the respectiveInstitutional Review Boards (IRB). Eligibility requirements includedmetastatic cancer expressing CEA and adequate hematologic, renal, andhepatic function. Trial participants were required to have receivedtreatment with standard therapy known to have a possible overallsurvival benefit or refused such therapy. Exclusion criteria includedchemotherapy or radiation within the prior 4 weeks, history ofautoimmune disease, viral hepatitis, HIV, or use of immunosuppressives.Patients who had been receiving bevacizumab or cetuximab for at least 3months prior to enrollment were permitted to continue receiving theseantibodies. Prior CEA immunotherapy was permitted. The study employed astandard 3+3 dose escalation strategy with dose limiting toxicities(DLT) defined as grade 3 or 4 major organ toxicity. The Ad5 [E1-,E2b-]-CEA(6D) doses were delivered to patients as follows: cohort 1:dose of 1×10⁹ VP in 0.5 ml subcutaneously (SQ) in the same thigh every 3weeks for 3 immunizations; cohort 2: dose of 1×10¹⁰ VP in 0.5 ml SQevery 3 weeks for 3 treatments; cohort 3: dose of 1×10¹¹ in 0.5 ml SQevery 3 weeks for 3 treatments. Following establishment of the dose of1×10¹¹ VP as safe, an additional 12 patients received Ad5 [E1-,E2b-]-CEA(6D) at this dose and schedule (phase II cohort). Aftercompleting the phase II cohort, an additional cohort (cohort 5) of six(6) patients received a dose of 5×10¹¹ VP in 2.5 ml SQ every 3 weeks for3 treatments to determine safety of the highest achievable dose. PMBCwere collected from patients just prior to the immunizations at weeks 0,3, 6, and three weeks following the last treatment. The PBMC were frozenin liquid nitrogen until ELISpot assays were performed. In cohort 5,fresh PBMC were analyzed in preliminary flow cytometry assays forpolyfunctional CD8+T lymphocytes.

Assessment of Clinical Activity.

Clinical activity was assessed according to Response Evaluation Criteriain Solid Tumors (RECIST 1.0 criteria) using computed tomography (CT) ormagnetic resonance imaging (MRI) scans obtained at baseline and aftertreatments were completed. Toxicity was assessed according to theNational Cancer Institute Common Terminology Criteria for Adverse Events(CTCAE) version 4. Peripheral blood CEA levels, hematology, serumchemistries, and anti-nuclear antibody titers were compared at baselineand 3 weeks following the final treatment. Survival was measured fromthe day of the first immunization until death from any cause.

Analysis of CMI Responses by ELISpot Assay.

An ELISpot assay for IFN-γ secreting lymphocytes was adapted from ourprevious animal studies and performed as described (Gabitzsch et al.(2010) Anti-tumor immunity despite immunity to adenovirus using a noveladenoviral vector Ad5 [E1-, E2b-]-CEA. Cancer Immunol Immunoother59:1131-1135). Briefly, isolated PBMCs (2×10⁵ cells/well) fromindividual patient samples were incubated 36-40 hours with a CEA peptidepool (15mers with 11aa overlap covering full length CEA with the 6Dmodification; 0.1 m/well) to stimulate IFN-γ producing T cells. CMIresponses to Ad5 were determined after exposure of patient PBMC toAd5-null (empty vector). Cells stimulated with concanavalin A (Con A) ata concentration of 0.25 μg/well served as positive controls. Coloredspot-forming cells (SFC) were counted using an Immunospot ELISpot platereader (Cellular Technology, Shaker Heights, Ohio) and responses wereconsidered to be positive if 50 SFC were detected/106 cells aftersubtraction of the negative control and SFC were ≧2-fold higher thanthose in the negative control wells.

Determination of Ad5 Neutralizing Antibody (NAb) Titers.

Endpoint Ad5 NAb titers were determined. Briefly, dilutions of heatinactivated test sera in 100 μL of DMEM containing 10% fetal calf serumwere mixed with 4×107 VP of Ad5 [E1-]-null and incubated for 60 minutesat room temperature. The samples were added to microwells containingHEK293 cells cultured in DMEM containing 10% heat inactivated calf serumat 2×103 cells/well for 24 hours at 37° C. in 5% CO2. The mixture wasincubated for an additional 72 hours at 37° C. in 5% CO2. An MTStetrazolium bioreduction assay (Promega Corp. Madison, Wis.) was used tomeasure cell killing and endpoint Ad5 NAb titers. Endpoint titers with avalue less than 1:25 were assigned a value of 0.

Statistics.

Statistical analyses comparing immune responses were performed employingthe Mann-Whitney test (PRISM, Graph Pad). Survival comparisons wereperformed employing Kaplan-Meier plots (PRISM, Graph Pad). Ad5 NAb titerand CEA-specific CMI were analyzed as continuous variables. Theassociation of Ad5 NAb titer with change in CEA-specific CMI was testedwith the Spearman correlation coefficient. The association of Ad5 NAbtiter with survival was tested with the Wald test of the proportionalhazards model. All tests used a 2-sided alpha of 0.05.

Demographics: All Patients

Thirty two patients with metastatic colorectal cancer, one with lungcancer and one with bladder cancer, median age 58 (range 38-77), who hadfailed a median of three prior chemotherapeutic regimens (range: 2->5),had a median performance status of 90% (range 70-100%), and had a medianof three sites of metastatic disease (range 1-5), were enrolled (Table2). The majority of patients was able to receive all threeimmunizations. Five patients who stopped immunizations prior tocompletion of all three treatments did so due to significant diseaseprogression.

TABLE 2 Patient Demographics ++

tient # Disease CEA CEA

/ Dose prior Mets # of status Survival Base- Week

ohort (VP) Dx Age Sex KPS CTx (# of sites) doses after tx (Months) line9

2/1 10⁹  C 67 M 70 >3 4 3 PD 3 (−) 98.8 867.4

3/1 10⁹  R 63 M 100 5 2 3 PD 9 (−) 195.1 472.2

4/1 10⁹  C 53 F 100 2 3 3 PD 11 (−)  65.4 196.8

5/2 10¹⁰ C 60 M 100 3 3 3 SD 12 (+)  2.5 3.7 (7 month follow-up)

7/2 10¹⁰ C 52 M 80 2 5 1 PD 1 (−) 120.7 Not Done

8/2 10¹⁰ C 42 F 100 3 3 3 PD 12 (+)  3.0 3.1

0/2 10¹⁰ C 58 M 90 3 3 3 PD 12 (−)  7.1 5.8

1/3 10¹¹ R 50 M 100 5 1 3 PD 12 (+)  21.0 25.9

2/3 10¹¹ C 48 M 100 1 2 3 PD 12 (+)  5.8 18.4

3/3 10¹¹ R 62 M 100 3 2 2 PD 4 (−) 172.9 Not Done

0/3 10¹¹ C 55 M 80 4 3 3 PD 12 (+)  3.2 11.5

5/3 10¹¹ C 58 F 80 3 4 3 PD 10 (−)  2.0 2.4

6/3 10¹¹ C 53 F 100 3 4 3 PD 6 (−) 6.1 12.7

7/3* 10¹¹ R 52 F 90 3 2 3 PD 3 (−) 204.8 Not Done

1/II 10¹¹ R 54 M 90 1 1 3 PD 12 (+)  17.1 96.4

2/II 10¹¹ C 66 F 80 1 2 2 PD 3 (−) 2549.5 Not Done

3/II 10¹¹ Bl 73 M 70 4 5 1 PD 0.25 (−)   Not Not Done Done

9/II 10¹¹ C 69 M 90 1 3 3 PD 12 (+)  264.3 638.0

0/II{circumflex over ( )} 10¹¹ C 59 M 100 5 4 3 SD 12 (+)  2.2 2.2

1/II{circumflex over ( )} 10¹¹ C 51 F 100 4 3 3 PD 12 (+)  2.0 2.7

6/II 10¹¹ C 77 F 80 2 2 3 PD 3 (−) 16.5 38.2

3/II 10¹¹ C 51 F 100 3 4 3 PD 4 (−) 32.4 211.4

4/II 10¹¹ C 57 M 90 3 3 3 PD 12 (+)  424.7 2073.6

7/II 10¹¹ R 58 M 90 2 2 3 PD 12 (+)  <0.5 0.6

8/II 10¹¹ L 67 M 100 2 0 3 Unknown 12 (+)  109.2 Not Done

4/II 10¹¹ C 67 M 90 2 3 3 PD 12 (+)  7.8 6.4

5/II 10¹¹ C 62 F 100 2 4 3 PD 7 (−) 391.2 Not Done

6/II 10¹¹ C 53 M 100 3 2 2 PD 4 (−) 4057.5 7859.1 (treatment #2)

0/5 5 × 10¹¹ C 38 M 90 4 3 3 PD 8 (+) 9.2 18.7

1/5 5 × 10¹¹ R 72 F 90 4 2 3 SD 7 (+) 3.9 5.6

2/5 5 × 10¹¹ R 53 M 90 4 3 3 PD 6 (−) 31.9 75.4

3/5 5 × 10¹¹ R 48 F 90 >3 2 3 PD 5 (−) 21.3 21.1

4/5 5 × 10¹¹ C 62 M 100 5 4 3 PD 6 (+) 1.9 2.4

5/5 5 × 10¹¹ C 60 F 90 3 5 2 PD 2 (−) 9.5 Not Done Dx = diagnosis (Bl =bladder cancer; C = colon cancer; L = lung cancer; R = rectal cancer)KPS = Karnofsky Performance Status *concurrent cetuximab; {circumflexover ( )}concurrent bevacizumab; @ concurrent panitumumab ++ Representsdisease status at 9 weeks post-initiation of immunizations PD =Progressive Disease; SD = Stable Disease (+) Alive; (−) Dead at lastfollow-up; survival rounded off to nearest month

indicates data missing or illegible when filed

Demographics: Colorectal Adenocarcinoma Patients

Thirty two patients, median age 57.5 (range 38-77) with metastaticcolorectal cancer, who had failed a median of three priorchemotherapeutic regimens (range: 2->5), had a median performance statusof 90% (range 70-100%), and had a median of three sites of metastaticdisease (range 1-5), were enrolled (Table 2). The majority was able toreceive all three immunizations. Four patients who stopped immunizationsearly did so due to significant disease progression. The colorectaladenocarcinoma patient demographics, albeit limited in size, comparesfavorably with previously published studies of patients withchemotherapy-refractory colorectal cancer (3-5).

Adverse Effects

A total of 94 immunization treatments were administered to all patients.There was no dose limiting toxicity and no serious adverse events thatresulted in treatment discontinuation at any vaccine dose level. Themost common toxicity (Table 3) was a self-limited, injection sitereaction. Other reactions that occurred at a low frequency includefever, flu-like symptoms, anorexia, chills, nausea, and headache. Thesesymptoms were also self-limiting and did not require intervention otherthan symptomatic measures such as acetaminophen.

Summary of Hematology, Chemistry, and ANA Values Pre and Post Treatment

Biological effects of ETBX-011 injections were monitored by recordingblood hematology, chemistry, and anti-nuclear antibody (ANA) values ofindividual patients in case record forms (CRFs). Of 34 total patientsentered into the trial, 28 received all three treatments with ETBX-011.For the 28 patients which received all three treatments, the bloodhematology, chemistry, and ANA values at week 0 (prior to firsttreatment) were compared with those obtained at week 9 (three weeksafter the third treatment). As shown in Table 4 below, there were nosignificant changes in chemistry or ANA values after treatments withETBX-011. There was only one significant change in the blood hematologyvalues. The basophil count was significantly (P=0.0403) lower at week 9after treatments. However, this value remained in the normal range forbasophil counts and, overall, there appeared to be no significantbiological effects.

Clinical Outcomes

CEA levels at baseline and week 9 were assessed in patients. Among thosewith CEA levels available at baseline and follow-up, three (patients010, 020, and 024) had no increase in CEA levels at the end of theimmunization period while the remaining patients showed increased CEAlevels. There were three patients with stable disease who remained soduring the 9 week study period. All other patients experienced somelevel of progressive disease (Table 2). Of the seven patients in cohorts1 and 2, there were five deaths and two patients remained alive at 12months following the initiation of immunization. Of the 21 patients incohort 3 and phase II, there were 10 deaths and all the remaining 11patients were alive at 12 months, respectively. Of the six patients incohort 5, there were three deaths and three patients were alive at 6, 7,and 8 months, respectively.

Of the 34 patients enrolled into the study, two patients received onetreatment, four patients received two immunization treatments, and theremaining 28 patients received all three immunization treatments. Allpatients were followed for survival and Kaplan-Meier plots and survivalproportions performed (PRISM software). Patient deaths were determinedby information gathered from the social security death index (SSDI)database and clinical charts.

The seven patients in cohorts 1 and 2 experienced a 12-month survivalproportion of 29% (FIG. 12A). Of the patients in cohorts 1 and 2,patient 004 survived 11 months and received additional post-immunizationtreatments with bevacizumab, folfox, and xeloda. Patient 003 survivednine months and received irradiation treatment after ETBX-011immunizations. Patient 005, alive at 12+ months, received irradiationtreatment and entered another clinical trial after immunizations.Patient 010 survived up to 12 months and entered two clinical trialsafter immunizations. Patients 002, 007, and 008 received no furthertreatments after immunizations and survived 3, 1, and 12+ months,respectively.

The 21 patients in cohort 3 and phase II experienced a 12-month survivalproportion of 48% (FIG. 12B). Of the patients in cohort 3 and phase II,one patient (017) received concurrent cetuximab during immunizations.Patients 020 and 021 received concurrent bevacizumab duringimmunizations. Patient 011 surviving over 12 months received radiationtreatment after ETBX-011 immunizations. Patients 012 and 016 survivedover 12 months and 6 months, respectively, and received additionalchemotherapy treatment after immunizations. Patient 013 survived 4months and received treatment with nexavar after immunizations. Patient015 survived 10 months and received follow-on treatment with cetuximab.Patient 019 survived over 12 months and received treatment withbevacizumab and xeliri after protocol immunizations. Patient 020survived over 12 months and received treatment with bevacizumab afterimmunizations. Patient 021 survived over 12 months and receivedfollow-on treatment with bevacizumab and xeloda. Patient 500 survivedover 12 months and received treatment with cetuximab and xeloda andentered a clinical trial after immunizations. Patient 501 survived over12 months and received treatment with cetuximab and irinotecan afterETBX-011 immunizations. Patient 508 has survived over 12 months;however, we have been unable to obtain further data on thecharacteristics of this patient. Patients 017, 023, 024, 025, 026, 502,504, 506, and 507 received no further treatment after immunizations andsurvived 3, 4, 12+, 7, 4, 3, 12+, 3, and 12+ months, respectively (+means still alive at the time of writing).

The six patients in cohort 5 experienced a 12-month survival proportionof 50% (FIG. 12C). Of the patients in this cohort, one patient (030) iscurrently alive at 8 months and received treatment with pazopanib andthreshold 302 chemotherapy after ETBX-011 immunizations. Patient 031 iscurrently alive at 7 months and has not received further treatment afterimmunizations. Patient 032 received concurrent panitumumab, survived 6months, and received treatment with folfox after immunizations. Patient033 received concurrent panitumumab, survived 5 months with noadditional therapy after immunizations. Patient 034 is currently aliveat 6+ months and received radiation and treatment with xeloda afterimmunizations. Patient 035 received two treatments and survived 2months.

With a median survival of 7.4 months, all 34 patients as a group(cohorts 1, 2, 3/phase II, and cohort 5) experienced a 12-month survivalproportion of 41% (FIG. 12D). Of the 34 patients entered in to thestudy, 28 patients received the three immunization treatments andexperienced a 12-month survival proportion of 55% (FIG. 13A) with amedian survival of 10.625 months. For the colorectal adenocarcinomapatients, 27 patients received the three immunization treatments andexperienced a 12-month survival proportion of 53% (FIG. 13B) with amedian survival of 10.00 months.

Evaluation of Immune Parameters in Treated Metastatic Colorectal CancerPatients

A secondary objective was to evaluate CEA specific immune responsesfollowing immunization treatments with the product. As determined byELISA, we observed no antibody activity directed against CEA. Weassessed CMI responses in colorectal cancer patients treated in cohort1, cohort 2, cohort 3/Phase II, and cohort 5. PBMCs were isolated priorto ETBX-011 treatment and after all treatments as well as three weeksfollowing the last treatment from patients. CEA specific ELISpot assayswere performed on PBMC as previously described (6) to determine thenumbers of interferon gamma (IFN-γ) secreting lymphocytes after exposureto CEA peptides in vitro. We determined the highest CMI responses duringimmunizations, regardless of time point (weeks 3, 6, or 9) in thepatients treated in cohort 1, cohort 2, cohort 3/Phase II, and cohort 5.As shown in FIG. 14, this analysis revealed a dose response toincreasing levels of product. The highest CMI levels occurred inpatients that received the highest dose of 5×10¹¹ VP (Cohort 5).

Determination of Induced CMI Responses to CEA.

ELISpot analysis was performed on cryopreserved PBMC samples drawnbefore each immunization and after completion of the final immunizationto assess CEA-specific CMI responses. We observed a dose response effectwith the highest magnitude CEA-specific CMI responses occurring inpatients who received the highest dose of Ad5 [E1-, E2b-]-CEA(6D) (FIG.14). Of the doses received, 0/3 (0%) patients in cohort 1 exhibitedpositive CEA-directed CMI responses, 1/4 (25%) patients in cohort 2exhibited positive CEA-directed CMI responses, 10/19 (53%) patients incohort 3/phase II exhibited positive CEA-directed CMI responses, and 4/6(67%) patients in cohort 5 exhibited positive CEA-directed CMIresponses. The time course of induction of CEA-specific CMI (FIG. 15)demonstrated that there may be plateau in the magnitude of CEA CMI priorto the last dose. In the largest group of patients who received the samedose (cohort 3 plus phase II), we observed a significant increase overbaseline in the average CEA-directed CMI responses at the 6 weekevaluation (P<0.05, Mann-Whitney test), averaging 94 SFC/106 PBMC, whichincreased further by the 9 week evaluation (FIG. 15). One patient(patient ID 13) had a highly elevated baseline CEA-specific immuneresponse (1100 SFC) and had elevated CMI at week six (2305 SFC) but didnot return for 9-week evaluation and therefore, was not included in CEACMI data analysis.

We also measured Ad5 NAb and CMI against Ad5 and correlated it withCEA-specific CMI. Each patient had their serum and PBMC sample tested atbaseline (prior to treatment) and at 9 weeks after completion of 3treatments. Nineteen of 31 colorectal cancer patients (61%) tested inthis study had Ad5 neutralizing activity in serum samples prior to theonset of treatment with ETBX-011. The mean pre-treatment Ad5 NAb titervalue obtained among all patients was 1:189±1:71 SEM (geometric mean1:21) and the mean pre-treatment Ad5 NAb titer among seropositivepatients was 1:308±1:108 (geometric mean 1:146). Analysis of serumsamples from patients who received 3 immunizations revealed Ad5 NAbtiters that were significantly increased (P<0.0001, Mann-Whitney test)by week 9 (mean 1:4767±1:1225 SEM (geometric mean 1:1541) when comparedwith their respective baseline values (FIG. 16). Analysis of PBMC forCMI responses to Ad5 also revealed a significant increase (P<0.01,Mann-Whitney test) in Ad5 directed CMI responses after immunizationswith ETBX-011 (FIG. 16).

Comparison of week 9 CEA-directed CMI responses from patients with lowbaseline pre-existing Ad5 immunity (Ad5 NAb≧200) verses those with highbaseline Ad5 immunity (Ad5 NAb >200) revealed no significant differencein immune responses (P>0.4, Mann Whitney test) (FIG. 17). Further, whenthe highest CEA specific CMI responses were compared with pre-existingor vector induced Ad5 NAb activity, there was no correlation betweenlevels of CEA CMI and Ad5 NAb activity (FIG. 17). These data indicatethat immunizations with ETBX-011 were not only able to overcomeself-tolerance, but were also able to induce CEA-specific immuneresponses in colorectal cancer patients despite the presence ofpre-existing and/or immunization induced Ad5 immunity. Together theseclinical trial data support the advancement to a Phase III clinicaltrial with overall survival as the primary endpoint

DISCUSSION

Adenoviral vectors have significant potential for use as cancertherapeutic vaccines because of their propensity to induce robustadaptive immune responses specifically against transgene products ingeneral. However, recombinant first generation Ad5 [E1-] vectors used inhomologous prime/boost regimens have been greatly limited in theirpotential efficacy due to the presence of pre-existing Ad5 immunity aswell as vector induced immunity. Specifically, Ad5-directed immunitymitigates immune responses to TAA that have been incorporated intoearlier generation Ad5 [E1-] based platforms. The Ad5 [E1-, E2b-]platform utilized in the present study was intended to accommodate ahomologous prime-boost regimen, by avoiding presentation of antigensthat are the targets of pre-existing Ad5 immunity. Since CEA has beenidentified as one of the priority cancer antigens by the National CancerInstitute, we investigated this TAA as a transgene to be incorporatedinto the new Ad5 [E1-, E2b-] vector platform for use as a cancertherapeutic vaccine. CEA expression in adults is normally limited to lowlevels in the gastrointestinal epithelium, whereas, CEA isover-expressed in adenocarcinomas of the colon and rectum and in manybreast, lung, and pancreas cancers. We chose the HLA A2 restrictedCAP1(6D) modification of CEA because, compared with the wild type CAP1epitope, CAP1(6D) can enhance the sensitization of CTLs and has beenincluded in our recent CEA-based vaccine constructs. Although we did nottest for HLA type because we used full length CEA that is notHLA-restricted, A*0201 is the allele observed most frequently inCaucasians (allele frequency 0.2717) and is common in other populations.However, it is possible to test patients for HLA type and utilize therelationship between HLA type and clinical and/or CMI responses.

Previously, we tested multiple subcutaneous immunizations employingthree administrations of a single dose level (1×10¹⁰ VP) of this classof Ad5 vaccine expressing the TAA CEA, (Ad5 [E1-, E2b-]-CEA(6D)) in apre-clinical murine model of CEA expressing cancer. In mice withpre-existing Ad5 immunity, we demonstrated the induction of potent CEAdirected CMI responses that resulted in anti-tumor activity and notedthat these CMI and anti-tumor responses were significantly greater thanthose responses induced by a current generation Ad5 [E1-] based vectorvaccine. We have also demonstrated in additional animal models (bothcancer and infectious disease targeted) that multiple subcutaneousimmunizations with vaccines based on the new Ad5 [E1-, E2b-] platforminduce CMI responses that were superior to those of current generationAd5 [E1-] based vaccines, can overcome the barrier of Ad5 immunity, andcan be utilized in multiple immunization regimens requiring a generationof robust CMI responses. In our present report, the greatest magnitudeof CEA-directed CMI responses occurred in patients receiving the highestdose of the vector. We observed that a CEA-directed CMI response wasinduced in a dose-responsive manner despite the presence of pre-existingand/or vector induced Ad5 immunity. No CEA directed antibody responseswere observed either pre- or post-vaccination employing an ELISAtechnique. In a preliminary analysis (data not shown), we also observeda population of polyfunctional CD8+T cells (those that secrete more thanone cytokine when activated) after immunizations, a sign of greaterfunctionality of T cells induced by the vaccine. These data support theuse of the Ad5 [E1, E2b-]-CEA(6D) vector in homologous prime-boostregimens designed to induce and increase CEA-directed CMI responses inpatients with advanced colorectal adenocarcinoma, as well as any numberof other vaccine amenable diseases or applications.

We believe there are factors that contribute to the favorable activityof this new platform. As compared to earlier generation Ad5 [E1-]vectors containing deletion in the early 1 (E1) gene region, the Ad5[E1-, E2b-] vector platform with additional deletions in the early 2b(E2b) gene region exhibits significantly reduced inflammatory responsesdirected at the vector. This can result in longer transgene expressionand a reduction in elimination of transgene expressing cells (e.g.,antigen presenting cells) that would otherwise occur due to inducedinflammatory responses. Since Ad5 late gene antigen expression issignificantly reduced as compared to earlier generation Ad5 platforms,this could enable the Ad5 [E1-, E2b-] platform to evade Ad5 immunemediated neutralizing activity for significantly longer periods of timeresulting in greater longevity and amplification of TAA expression. Inaddition, a E2b gene product, a polymerase, is a known target of humancellular memory immune responses to Ad5 infection and its eliminationfrom the vaccine could be furthering its capability in the setting ofpre-existing Ad5 immunity. Without being bound by theory, the extendedand/or greater expression of TAA by the vector in this milieu couldresult in a more effective immune response against the target antigen.However, it is also possible that this vector configuration producesbetter transgene expression, different biodistribution, or differentinnate/adaptive immune effects that impact the effectiveness of thisvector, rather than escape from pre-existing immunity.

Of interest is the observation that treated patients in our studyexhibited favorable survival probability. Overall, all 25 patientstreated at least 2 times with Ad5 [E1-, E2b-]-CEA(6D) exhibited a12-month survival probability of 48% and this was achieved despite thepresence of significant levels of pre-existing Ad5 neutralizing antibodytiters.

In other clinical trials, immunotherapeutic agents have been found toincrease overall survival without having a direct impact on time toobjective disease progression, a trend noted in our study as well.Without being bound by theory, by engaging the patient's immune system,active immunotherapeutics, such as Ad5 [E1-, E2b-]-CEA(6D), could inducecontinuous immunologic anti-tumor responses over a long period of timethat could result in a “deceleration” or alteration in specific aspectsof the rapid growth rate or spread of the tumor not measured by standardresponse assessments. Indeed, we have observed slower tumor progressionin Ad5 immune mice harboring established CEA-expressing tumors followingtreatment with Ad5 [E1-, E2b-]-CEA(6D). Moreover, it has been noted thatoverall survival might be the only true parameter for determination ofclinical efficacy of any potential cancer (immune) therapy.

As with any new treatment modality, safety is an important factor. Inthis Phase I/II trial, we demonstrated that the Ad5 [E1-, E2b-]-CEA(6D)could be manufactured to scale, as well be easily and repeatedlyadministered by conventional subcutaneous injection techniques. The mostcommon adverse effects were site of injection reactions and flu-likesymptoms consisting of fever, chills, headache, and nausea. There was noimpact on blood hematology or serum chemistries and, overall, thetreatments were well tolerated. Specifically, no SAE were noted, and notreatments were stopped due to adverse events, indicating that a doselimitation to use of Ad5 [E1-, E2b-]-CEA(6D) in this clinicalapplication had not been met.

These data suggest that patients with advanced colorectal cancer whichare treated with Ad5 [E1-, E2b-]-CEA(6D) do not have serious adverseeffects and may experience extension of life even if they havepre-existing immunity to Ad5. The results of this trial are encouragingenough to advance to a large, randomized, single agent trial. Theobservation that some of the patients experienced an increase of CMIwhich is dose dependent, could be an indication that this may play arole in their clinical outcome.

TABLE 3 Adverse Events % Incidence # Of Unrelated/ Probably/ *Grade (G1,(Based on 94 Adverse Events Events Unlikely Possible Definite G2, or G3)treatments) Injection Site Reaction 21 21 G1 (19): G2 (2) 22.3 Pain (alltypes) 17 17 G1 (8); G2 (7); 18.1 G3 (2) Fever 10 4 2 4 G1 (7); G2 (3)10.6 Flu-like symptoms 10 3 5 2 G1 (9); G2 (1) 10.6 Fatigue 8 6 2 G1(5); G2 (2); 8.5 G3 (1) Shortness of Breath 6 6 G1 (3); G2 (3) 6.4Anorexia 5 4 1 G1 (3); G2 (2) 5.3 Chills 5 1 1 3 G1 (5) 5.3 Nausea 5 4 1G1 (5) 5.3 Constipation 5 5 G1 (3); G2 (2) 5.3 Edema 5 5 G1 (3); G2 (2)5.3 Vomiting 4 4 G1 (4) 4.3 Hypertension 3 3 G1 (2); G2 (1) 3.2 Anemia 33 G1 (1); G2 (1); 3.2 G3 (1) Cough 2 2 G1 (2) 2.1 Depression 2 2 G1 (2)2.1 Diarrhea 2 2 G1 (2) 2.1 Headache 2 1 1 G1 (2) 2.1 Hypoalbuminemia 22 G1 (1); G2 (1) 2.1 Hypokalemia 2 2 G1 (1); G2 (1) 2.1 Pleural Effusion2 2 G2 (1); G3 (1) 2.1 Alkaline Phosphatase Increase 2 2 G1 (1); G3 (1)2.1 Myalgia 2 2 G1 (2) 2.1 Night Sweats 2 2 G1 (1); G2 (1) 2.1 Sleep 2 2G1 (2) 2.1 Low Magnesium 2 2 G1 (2) 2.1 Abdominal Bloating 1 1 G1 (1)1.1 Abdominal Distention 1 1 G3 (1) 1.1 Abdominal Swelling 1 1 G2 (1)1.1 Abdominal Wound 1 1 G2 (1) 1.1 ALT Increase 1 1 G1 (1) 1.1 ASTIncrease 1 1 G2 (1) 1.1 Biliary Obstruction 1 1 G3 (1) 1.1 BowelObstruction 1 1 G3 (1) 1.1 Cold 1 1 G1 (1) 1.1 Dyspnea 1 1 G3 (1) 1.1Dysuria 1 1 G1 (1) 1.1 Frequent Urination 1 1 G1 (1) 1.1 GI Disorder 1 1G3 (1) 1.1 Extra Pyramidial Movements 1 1 G1 (1) 1.1 Insomnia 1 1 G1 (1)1.1 Herpes Simplex 1 1 G1 (1) 1.1 Hypotension 1 1 G1 (1) 1.1 Loss ofAppetite 1 1 G1 (1) 1.1 Low White Blood Cells 1 1 G1 (1) 1.1Numbness/Sensation in Fingertips 1 1 G1 (1) 1.1 Onset of Menses 1 1 G1(1) 1.1 Poor Quality Sleep 1 1 G1 (1) 1.1 Presyncope 1 1 G2 (1) 1.1Pruritis 1 1 G1 (1) 1.1 Rash-Right Lower Eye Lid 1 1 G1 (1) 1.1Red/Swelling Right Upper Eyelid 1 1 G1 (1) 1.1 Renal Calculi 1 1 G2 (1)1.1 Runny Nose 1 1 G1 (1) 1.1 Shallow Breathing 1 1 G1 (1) 1.1 Skin Rash1 1 G1 (1) 1.1 Vaginal Discharge 1 1 G1 (1) 1.1 Concentration 1 G1 (1)1.1 Weight Loss 1 1 G2 (1) 1.1 Arthritis Joint Inflammation 1 1 G1 (1)1.1 Flushing 1 1 G1 (1) 1.1 Acute Renal Failure Disease G3 (1) 1.1progression *Parenthesis ( ) indicates numbers of events.

TABLE 4 Hematology, Chemistry, and ANA values Week 0 value Week 9 value(Mean ± SEM) (Mean ± SEM) Hematology Test Hgb (g/dL) 13.09 ± 0.313 12.48± 0.413 Hct (%) 39.63 ± 0.875 37.92 ± 1.140 Plts (×109/L) 225.1 ± 20.76247.3 ± 23.57 WBC (×103/mm3)  6.81 ± 0.532  8.21 ± 0.741 Neutrophils (%)64.46 ± 2.068 67.28 ± 3.268 Lymphocytes (%) 23.23 ± 1.874 18.34 ± 2.071Monocytes (%)  8.86 ± 0.462  7.68 ± 0.569 Eosinophils (%)  3.97 ± 0.677 3.16 ± 0.685 Basophils (%)  0.52 ± 0.056  0.38 ± 0.048 Chemistry TestNa (mEq/L) 139.2 ± 0.424 137.9 ± 0.718 K (mEq/L)  3.90 ± 0.085  3.80 ±0.073 Cl (mEq/L) 105.0 ± 0.561 103.3 ± 1.061 CO2 (mEq/L)  27.8 ± 0.37427.63 ± 0.458 BUN (mg/dL)  17.0 ± 1.136  17.1 ± 1.611 Creatinine (mg/dL) 0.81 ± 0.046  0.86 ± 0.054 Glucose (mg/dL) 121.8 ± 7.458 123.5 ± 7.885Ca (mg/dL)  8.84 ± 0.075  8.87 ± 0.073 Total protein (g/dL)  6.95 ±0.078  6.67 ± 0.100 Albumin (g/dL)  3.78 ± 0.085  3.62 ± 0.113 AST (U/L)31.71 ± 3.846 31.88 ± 3.506 ALT (U/L) 27.83 ± 4.228 25.67 ± 3.414Alkaline phosphatase (U/L) 107.0 ± 13.30 124.0 ± 17.40 Bilirubin (mg/dL) 0.78 ± 0.071  0.75 ± 0.079 *ANA Test Titer 103.3 ± 51.04 123.3 ± 50.56*Values represent inverse of the titer and are from patients withpositive values.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety to the same extentas if each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Aspects of the embodiments can be modified, if necessary to employconcepts of the various patents, application and publications to provideyet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1.-252. (canceled)
 253. A composition comprising a recombinant nucleicacid vector comprising a sequence with at least 80% sequence identity toSEQ ID NO:
 3. 254. The composition of claim 253, wherein the recombinantnucleic acid vector comprises a region with at least 80% sequenceidentity to a region of SEQ ID NO: 3 selected from the group consistingof nucleic acids 26048-26177 of SEQ ID NO: 3, nucleic acids 26063-26141of SEQ ID NO: 3, nucleic acids 1-103 of SEQ ID NO: 3, nucleic acids54-103 of SEQ ID NO: 3, nucleic acids 32214-32315 of SEQ ID NO: 3, andnucleic acids 32214-32262 of SEQ ID NO:
 3. 255. The composition of claim253, wherein the recombinant nucleic acid vector comprises a regionencoding a peptide with at least 80% sequence identity to an amino acidsequence encoded by nucleic acids 1057-3165 of SEQ ID NO:
 3. 256. Thecomposition of claim 253, wherein the recombinant nucleic acid vectorcomprises a sequence with at least 90% sequence identity to SEQ ID NO:3.
 257. A composition comprising a recombinant nucleic acid vectorcomprising a modified carcinoembryonic antigen (CEA) sequence encoding amodified CEA peptide; wherein the modified CEA sequence comprises asequence with at least 80% sequence identity to SEQ ID NO: 2; andwherein the recombinant nucleic acid vector is a replication defectiveadenovirus vector.
 258. The composition of claim 257, whereinreplication defective adenovirus vector comprises a replicationdefective adenovirus serotype-5 (Ad5) vector with a deletion in an early1 (E1) gene region and a deletion in an early 2b (E2b) gene region. 259.The composition of claim 257, wherein the modified CEA sequencecomprises a sequence with at least 90% sequence identity to SEQ ID NO:2.
 260. A composition comprising a nucleic acid vector comprising asequence encoding a modified carcinoembryonic antigen (CEA) peptide;wherein the modified CEA peptide comprises a modification of from 1 to25 amino acids; and wherein the recombinant nucleic acid vectorcomprises a replication defective adenovirus serotype-5 (Ad5) vectorwith a deletion in an early 1 (E1) gene region and a deletion in anearly 2b (E2b) gene region.
 261. The composition of claim 260, whereincells transfected with the recombinant nucleic acid vector overexpressthe modified CEA peptide.
 262. The composition of claim 260, wherein therecombinant nucleic acid vector induces an immune response in a subjectto the modified CEA peptide.
 263. A method of treatment comprising: (a)performing a first treatment to a human, the first treatment comprisingadministering to the human for a total of 3 times at 3-4 week intervals:a first replication defective adenovirus serotype-5 (Ad5) vector with adeletion in an early 1 (E1) gene region, a deletion in an early 2b (E2b)gene region, and a sequence that encodes a modified carcinoembryonicantigen (CEA) peptide, wherein the recombinant nucleic acid vectorinduces an immune response to the modified CEA peptide; and (b)performing one or more additional treatments to the human, the one ormore additional treatments comprising administering to the human at 2-4month intervals starting about 2 months after the first treatment: asecond replication defective Ad5 vector with a deletion in an E1 generegion, a deletion in an E2b gene region, and a sequence that encodes amodified CEA peptide, wherein the recombinant nucleic acid vectorinduces an immune response to the modified CEA peptide.
 264. The methodof claim 263, wherein the first replication defective adenovirus vectorand the second replication defective adenovirus vector are the same.265. The method of claim 263, wherein the administering of (a) or (b)comprises administering at least 1×10¹¹ replication defective adenoviralparticles.
 266. The method of claim 263, wherein the human has tumorcells that overexpress CEA.
 267. The method of claim 263, wherein thehuman has pre-existing immunity to Ad5.
 268. The method of claim 263,wherein the human is not concurrently being treated with steroids,corticosteroids, immunosuppressive agents, immunotherapy, or anycombination thereof.
 269. The method of claim 263, wherein the human isnot being treated with steroids, corticosteroids, immunosuppressiveagents, immunotherapy, or any combination thereof, prior to theadministering of (a).
 270. The method of claim 263, wherein the human isnot concurrently being treated with cytotoxic chemotherapy.
 271. Themethod of claim 263, wherein the human is not concurrently being treatedwith radiation therapy.
 272. The method of claim 263, wherein the humandoes not have an autoimmune disease.
 273. The method of claim 263,wherein the human does not have inflammatory bowel disease, systemiclupus erythematosus, ankylosing spondylitis, scleroderma, autoimmunethyroid disease, vitiligo, multiple sclerosis, viral hepatitis, or HIV.